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Heat shock genes and proteins from neisseria meningitidis, candida glabrata and aspergillus fumigatus

Title: Heat shock genes and proteins from neisseria meningitidis, candida glabrata and aspergillus fumigatus

Related Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Antigen, Epitope, Or Other Immunospecific Immunoeffector (e.g., Immunospecific Vaccine, Immunospecific Stimulator Of Cell-mediated Immunity, Immunospecific Tolerogen, Immunospecific Immunosuppressor, Etc.), Bacterium Or Component Thereof Or Substance Produced By Said Bacterium (e.g., Legionella, Borrelia, Anaplasma, Shigella, Etc.), Neisseria (e.g., Neisseria Gonorrhoeae, Etc.), Neisseria Meningitidis

Heat shock genes and proteins from neisseria meningitidis, candida glabrata and aspergillus fumigatus description/claims

The Patent Description & Claims data below is from USPTO Patent Application 20070122434, Heat shock genes and proteins from neisseria meningitidis, candida glabrata and aspergillus fumigatus.

Brief Patent Description - Full Patent Description - Patent Application Claims

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No. 10/269,557, filed Oct. 11, 2002, which is a divisional of U.S. application Ser. No. 09/207,388, filed Dec. 8, 1998, now U.S. Pat. No. 6,497,880. The entire content of the prior application is incorporated by reference in its entirety.

TECHNICAL FIELD OF THE INVENTION

[0002] This invention relates to heat shock proteins of the Hsp60 family from Candida glabrata and Aspergillus fumigatus and a heat shock protein of the Hsp70 family from Neisseria meningitidis, including fragments thereof, and uses of such proteins and nucleic acid molecules encoding these proteins.

BACKGROUND OF THE INVENTION

[0003] Meningitis is an infection of the fluid of the spinal cord and the fluid that surrounds the brain. The disease is caused either by a viral or a bacterial infection. Viral meningitis is typically less severe than bacterial meningitis and resolves without specific treatment. In contrast, bacterial meningitis can be rather severe and can cause brain damage, hearing loss or learning disability. Symptoms of meningitis are high fever, headache and stiff neck. These symptoms may develop over a span of several hours, or may take 1-2 days. Other symptoms may be nausea, vomiting, discomfort looking into bright light, confusion or sleepiness. In young children, the classical symptoms may be absent or may not be easily detected, and the child may appear to be slow, inactive, irritable, vomiting or feeding poorly. As the disease progresses, seizures may occur.

[0004] Bacterial meningitis may be caused by Haemophilus influenzae, Streptococcus pneumoniae or Neisseria meningitidis. Because all children (in the U.S.) are now given a vaccine against Haemophilus influenzae in the course of their routine immunizations, meningitis due to this organism is now relatively uncommon. Thus, the major bacterial disease-causing agents are now Streptococcus pneumoniae and Neisseria meningitidis.

[0005] Early diagnosis and treatment are critically important. It must be determined whether symptoms are due to a viral or bacterial agents, and, if they are caused by a bacterial agent, which bacterium is involved. Present methods of diagnosis are relatively slow. They involve obtaining spinal fluid by performing a spinal tap. Bacteria are identified by cultivation of spinal fluid.

[0006] Bacterially caused meningitis can be treated by antibiotics. However, it is critically important that treatment commence early in the course of the disease. Obviously, antibiotic therapy may be jeopardized by the development of antibiotic-resistant strains of disease-causing bacteria. Because of this concern, and also because of cost-benefit considerations, vaccination against the bacteria causing the disease would be preferable, at least in regions, in which the disease is endemic. As discussed before, U.S. children are routinely vaccinated against Haemophilus influenzae, but not against Neisseria meningitidis and Streptococcus pneumoniae. It is noted that vaccines against the latter two organisms have been generated. One such vaccine protects against four strains of Neisseria meningitidis. However, the vaccine appears not to be effective in children under 18 months of age. Similarly, a vaccine containing polysaccharide antigens for 14 of the 83 capsular types of Streptococcus pneumoniae was developed. The vaccine was found to be 57% effective in two large studies. As with the Neisseria vaccine, children under the age of two years do not appear to be protected by the vaccine. Thus, there is a need for improved vaccines against the latter two species of bacteria. The information provided here was obtained from publications by the Division of Bacterial and Mycotic Diseases of the National Center for Infectious Diseases, and the Centers for Disease Control and Prevention (www.cdc.gov/ncidod/dbmd/bactmen.htm; May 28, 1998), by Lonks and Medeiros, Antimicrobial Therapy 1 79:523-35, 1995, by Butler et al., JAMA 270:1826, 1993, and by Gotschlich et al., Antibodies in Human Diagnosis and Therapy 391-402 (Haber and Krause eds., 1977).

[0007] Aspergillosis is an opportunistic infection occurring in compromised individuals and is caused by molds of the genus Aspergillus, of which Aspergillus fumigatus is an important species. Aspergillus is ubiquitous and is distributed worldwide. Infection generally involves inhalation of fungal elements. Aspergillosis has several clinical manifestations, including colonization of the ear or the lungs, allergic pulmonary involvement, and invasive pulmonary and disseminated infections. The pulmonary invasive and disseminated forms of infections have a grave prognosis, including a high rate of mortality. Susceptible hosts include cancer patients, patients treated with immunosuppressive or cytotoxic drugs, and those otherwise debilitated such as AIDS patients, neutropenic cancer patients, or patients receiving adrenal corticosteroid drugs. Segal, Vaccines against Fungal Infections, CRC Crit. Rev. Microbiology 14:229, 1987. Rolston and Bodey, Infections in patients with cancer, Cancer Medicine (Holland et al. eds), 1997. The true incidence of aspergillosis is not known in the HIV/AIDS population, in part because the condition is frequently not diagnosed. However, it is clear that the incidence is increasing. Ampel has reported that more than 75 cases have been documented in the literature. Ampel, Emerging disease issues and fungal pathogens associated with HIV infection, Emerging Infectious Diseases 2:109-116, 1996. Aspergillosis has been reported to occur in 20-50% of patients with acute leukemia. It is noted that many cases of Aspergillus infection in cancer patients are not diagnosed until an autopsy is performed after death. Clearly, aspergillosis is becoming increasingly common among neutropenic patients and in cancer patients receiving corticosteroid drugs. Rolston and Bodey, supra. An article in 1992 by Bodey et al. reports on the incidence of fungal infections based on an international autopsy survey. Bodey et al., Fungal Infections In Cancer Patients, An International Autopsy Survey, Eur. J. Clin. Microbiol. Infect. Dis. 11:99-109, 1992. Countries included are Austria, Belgium, Canada, Germany, Italy, Japan, Netherlands and the UK. It was concluded that 25% of leukemia patients had fungal infections. Of these infected patients, 66% had candidiasis, and 34% aspergillosis. Estimates of rates of fungal infections in organ transplant patients as high as about 40% were published. Paya, Clin. Infect. Dis. 16:677-688, 1993. More than 80% of these infections were due to Candida and Aspergillus. As alluded to before, diagnosis of aspergillosis is not infrequently missed, and there is therefore a need for improved methods of diagnosis.

[0008] Candidiasis is a fungal infection caused by yeasts of the genus Candida. Among the more than 80 known species only seven species appear to be pathogenic. The major disease-causing species is Candida albicans. Among the pathogenic species is also found Candida glabrata, formerly known as Torulopsis glabrata. Clinical manifestations of Candida infection range from superficial cutaneous infections to disseminated disease. Infections range from acute to chronic. They can involve skin and nails, the mucosal membranes of the mouth and vagina, and various internal organs such as the lungs, gastrointestinal tract, and circulatory and central nervous systems. Manifestations can be oral thrush, vaginitis, balanitis, diaper rash, chronic mucocutaneous conditions, bronchitis or pneumonia, meningitis, endocarditis, and septicemia. While the superficial forms of candidiasis have been well known since antiquity, the incidence of the disseminated forms has increased recently, presumably because of the extensive use of antibiotics, corticosteroids, cytotoxic drugs, organ transplantation and other complex surgical procedures. It is important to note that today the majority of systemic or invasive fungal infections are due to Candida species. Segal, supra. Stringer, Mass. High Tech. 14:3, 1997.

[0009] Mortality from systemic candidiasis remains high, in the order of 38-59%. The mainstay for treatment is amphotericin B, and an alternative is fluconazole. In a multicenter trial, amphotericin B was 79% effective, and fluconazole 70%. Note that Candida glabrata is resistant to fluconazole. Because mortality remains high, an effective vaccine against candidiasis to be used in high-risk populations would be desirable.

[0010] Since Candida albicans is the major cause of candidiasis, essentially all work relating to both diagnosis and vaccination concerned this particular species. Thus, it is not clear to what extent diagnostic procedures developed and vaccination approaches taken would also detect or protect against other species such as Candida glabrata.

[0011] Therefore, there is a need in the art to identify and isolate novel stress proteins and nucleic acids encoding the same from Neisseria meningitidis, Candida glabrata and Aspergillus fumigatus, which are useful in the detection, diagnosis and treatment of infections caused by these organisms.

SUMMARY OF THE INVENTION

[0012] The present invention provides methods and compositions comprising isolated nucleic acid molecules specific to Neisseria meningitidis, Candida glabrata and Aspergillus fumigatus, as well as vector constructs and isolated polypeptides specific to Neisseria meningitidis, Candida glabrata and Aspergillus fumigatus. Such compositions and methods are useful for the diagnosis of infection and for generating an immune response to the respective organisms.

[0013] Thus, in one aspect the present invention provides an isolated nucleic acid molecule encoding a Neisseria meningitidis Hsp70. In some embodiments, the isolated nucleotide molecule is selected from the group consisting of: (a) an isolated nucleic acid molecule comprising the sequence of SEQ ID NO:1 (FIG. 4); (b) an isolated nucleic acid molecule comprising the sequence of SEQ ID NO:1 from nucleotides 358-2286; (c) an isolated nucleic acid molecule comprising the sequence of SEQ ID NO:3 (FIG. 6) from nucleotides 4-1932; (d) an isolated nucleic acid molecule comprising the sequence of SEQ ID NO:3 (FIG. 8); (e) an isolated nucleic acid molecule comprising the sequence of SEQ ID NO:4 (FIG. 9); (f) an isolated nucleic acid molecule complementary to any one of the nucleotides of SEQ ID NOS: 1, 3 and 4 set forth in (a) through (e).

[0014] In another aspect, the present invention provides an isolated nucleic acid molecule which is a variant of, or is substantially similar to, the Neisseria Hsp70 nucleotide molecules described above. In further aspects, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that is identical to a segment of contiguous nucleotide bases comprising at least 25% of SEQ ID NOS: 1, 3 or 4 or a complement thereof, or an isolated nucleic acid molecule encoding Hsp70 comprising a nucleic acid sequence that encodes a polypeptide comprising any one of SEQ ID NOS: 15 and 16 or a variant Hsp70 that is at least 95% homologous to a polypeptide according to any one of SEQ ID NOS: 15 and 16.

[0015] In one embodiment, the present invention provides an isolated nucleic acid molecule as described above, the molecule encoding a polypeptide that is able to be selectively bound by an antibody specific for a Neisseria meningitidis Hsp70.

[0016] In still another aspect, the present invention provides an isolated nucleic acid molecule encoding at least 8 contiguous amino acids of a Neisseria meningitidis Hsp70 polypeptide selected from amino acid residues of FIG. 6 (SEQ ID NO: 15), wherein the encoded Neisseria meningitidis Hsp70 polypeptide is able to bind to a major histocompatibility complex.

[0017] In still further aspects the present invention provides an isolated Neisseria meningitidis Hsp70 polypeptide.

[0018] In some embodiments, the isolated Hsp70 polypeptide comprises the amino acid sequence of FIG. 6 (SEQ ID NO:15), or variants thereof, preferably wherein the polypeptide is able to be selectively bound by an antibody specific for a Neisseria meningitidis Hsp70. In further embodiments, the isolated Hsp70 polypeptide is fused to an additional polypeptide to create a fusion protein.

[0019] In still yet further aspects the present invention provides an isolated Hsp70 polypeptide comprising at least 8 contiguous amino acids selected from amino acid residues of FIG. 6 (SEQ ID NO:15), wherein the Hsp70 polypeptide is capable of binding to a major histocompatibility complex and eliciting or enhancing an immune response to Neisseria meningitidis in a human being.

[0020] In certain embodiments, the isolated Hsp70 polypeptide is derived by proteolytic cleavage or chemical synthesis, or is an expression product of a transformed host cell containing a nucleic acid molecule encoding the Hsp70 or portion thereof. In further certain embodiments, the isolated Hsp70 polypeptide comprises greater than 95% homology to the Hsp70 polypeptide of FIG. 6 (SEQ ID NO:15), and the isolated Hsp70 polypeptide is able to be selectively bound by an antibody specific for a Neisseria meningitidis Hsp70.

[0021] In still yet another aspect the present invention provides an isolated polypeptide wherein the polypeptide is an expression product of a transformed host cell containing one of the aforementioned nucleic acid molecules derived from Neisseria.

[0022] In still yet further aspects the present invention provides vectors comprising at least one of the aforementioned nucleic acid molecules derived from Neisseria. In certain embodiments, the vector is an expression vector comprising a promoter in operative linkage with the isolated nucleic acid molecule encoding the Hsp70 or portion thereof, preferably further comprising a selectable or identifiable marker and/or wherein the promoter is a constitutive or an inducible promoter. The present invention also provides host cells containing such vectors. In certain embodiments, the host cell is selected from the group consisting of a bacterial cell, a mammalian cell, a yeast cell, a plant cell and an insect cell.

[0023] In still yet other aspects the present invention provides compositions comprising a Neisseria Hsp70 polypeptide in combination with a pharmaceutically acceptable carrier or diluent. In certain embodiments, the composition is suitable for systemic administration, oral administration, intranasal administration or parenteral administration.

[0024] In yet other aspects the present invention provides methods for eliciting or enhancing an immune response in a mammal against Neisseria, comprising administering to the mammal in an amount effective to elicit or enhance the response, a Neisseria Hsp70 polypeptide in combination with a pharmaceutically acceptable carrier or diluent; methods for eliciting or enhancing an immune response in a mammal to a polypeptide comprising administering to the mammal a fusion protein containing sequences of the polypeptide fused to the Neisseria Hsp70 polypeptide in combination with a pharmaceutically acceptable carrier or diluent; and methods for eliciting or enhancing an immune response in a mammal against a target antigen comprising administering to the mammal the target antigen joined to a Neisseria Hsp70 polypeptide in combination with a pharmaceutically acceptable carrier or diluent.

[0025] In still another aspect, this invention provides PCR primers and probes for detecting DNA encoding a Neisseria meningitidis. Hsp70 that includes at least about 15 contiguous bases from any one of SEQ ID NOS: 1, 3 and 4, or to compliment thereof. In a related aspect, the invention provides a method for diagnosing the presence of a Neisseria meningitidis in a subject sample that includes the steps of obtaining a DNA fraction from the subject sample; and performing a PCR amplification of the DNA fraction using at least one PCR primer that includes at least about 15 contiguous bases from any one of SEQ ID NOS: 1, 3 and 4, or a compliment thereof.

[0026] The present invention also provides an isolated nucleic acid molecule encoding a Aspergillus fumigatus Hsp60. In some embodiments, the isolated nucleotide molecule is selected from the group consisting of: (a) an isolated nucleic acid molecule comprising the sequence of SEQ ID NO:5 (FIG. 14); (b) an isolated nucleic acid molecule comprising the sequence of SEQ ID NO:5 from nucleotides 300-2234; (c) an isolated nucleic acid molecule comprising the sequence of SEQ ID NO:5 from nucleotides 300-410, nucleotides 514-655 and nucleotides 724-2234; (d) an isolated nucleic acid molecule comprising the sequence of SEQ ID NO:6 (FIG. 16); (e) an isolated nucleic acid molecule comprising the sequence of SEQ ID NO:7 (FIG. 18); (f) an isolated nucleic acid molecule complementary to any one of the nucleotides of SEQ ID NOS: 5 to 7 set forth in (a) through (e), respectively.

[0027] In another aspect, the present invention provides an isolated nucleic acid molecule that is a variant of, or is substantially similar to, the Aspergillus Hsp60 nucleotide molecules described above. In further aspects, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that is identical to a segment of contiguous nucleotide bases comprising at least 25% of SEQ ID NOS: 5 to 7 or a complement thereof or an isolated nucleic acid molecule encoding Hsp60 comprising a nucleic acid sequence that encodes a polypeptide comprising any one of SEQ ID NOS: 5, 6 or 7 or a variant Hsp60 that is at least 95% homologous to a polypeptide according to any one of SEQ ID NOS: 5, 6, or 7.

[0028] In one embodiment, the present invention provides an isolated nucleic acid molecule according as described above, the molecule encoding a polypeptide that is able to be selectively bound by an antibody specific for a Aspergillus fumigatus Hsp60.

[0029] In still another aspect in one aspect the present invention provides an isolated nucleic acid molecule encoding at least 8 contiguous amino acids of a Aspergillus fumigatus Hsp60 polypeptide selected from amino acid residues according to FIG. 14, wherein the encoded Aspergillus fumigatus Hsp60 polypeptide is able to bind to a major histocompatibility complex.

[0030] In still further aspects the present invention provides an isolated Aspergillus fumigatus Hsp60 polypeptide.

[0031] In some embodiments, the isolated Hsp60 polypeptide comprises the amino acid sequence of FIG. 14, or variants thereof, preferably wherein the polypeptide is able to be selectively bound by an antibody specific for a Aspergillus fumigatus Hsp60. In further embodiments, the isolated Hsp60 polypeptide is fused to an additional polypeptide to create a fusion protein.

[0032] In still yet further aspects the present invention provides an isolated Hsp60 polypeptide comprising at least 8 contiguous amino acids selected from amino acid residues according to FIG. 14, wherein the Hsp60 polypeptide is capable of binding to a major histocompatibility complex and eliciting or enhancing an immune response to Aspergillus fumigatus in a human being.

[0033] In certain embodiments, the isolated Hsp60 polypeptide is derived from proteolytic cleavage or chemical synthesis, or is an expression product of a transformed host cell containing a nucleic acid molecule encoding the Hsp60 or portion thereof. In certain further embodiments, the isolated Hsp60 polypeptide comprises greater than 95% homology to the Hsp60 polypeptide of FIG. 14, and the isolated Hsp60 polypeptide is able to be selectively bound by an antibody specific for a Aspergillus fumigatus Hsp60.

[0034] In still yet another aspect the present invention provides an isolated polypeptide wherein the polypeptide is an expression product of a transformed host cell containing at least one of the nucleic acid molecules derived from the aforementioned Aspergillus molecules.

[0035] In still yet further aspects the present invention provides vectors comprising at least one of the aforementioned nucleic acid molecules derived from Aspergillus. In certain embodiments, the vector is an expression vector comprising a promoter in operative linkage with the isolated nucleic acid molecule encoding the Hsp60 or portion thereof, preferably further comprising a selectable or identifiable marker and/or wherein the promoter is a constitutive or an inducible promoter. The present invention also provides host cells containing such vectors. In certain embodiments, the host cell is selected from the group consisting of a bacterial cell, a mammalian cell, a yeast cell, a plant cell and an insect cell.

[0036] In still yet other aspects the present invention provides compositions comprising an Aspergillus Hsp60 polypeptide in combination with a pharmaceutically acceptable carrier or diluent. In certain embodiments, the composition is suitable for systemic administration, oral administration, intranasal administration or parenteral administration.

[0037] In yet other aspects the present invention provides methods for eliciting or enhancing an immune response in a mammal against Aspergillus, comprising administering to the mammal in an amount effective to elicit or enhance the response, an Aspergilllus Hsp60 polypeptide in combination with a pharmaceutically acceptable carrier or diluent; methods for eliciting or enhancing an immune response in a mammal to a polypeptide comprising administering to the mammal a fusion protein containing sequences of the polypeptide fused to the Hsp60 polypeptide in combination with a pharmaceutically acceptable carrier or diluent; and methods for eliciting or enhancing an immune response in a mammal against a target antigen comprising administering to the mammal the target antigen joined to an Aspergillus Hsp60 polypeptide in combination with a pharmaceutically acceptable carrier or diluent.

[0038] In still another aspect, this invention provides PCR primers and probes for detecting DNA encoding a Aspergillus fumigatus Hsp60 that includes at least about 15 contiguous bases from any one of SEQ. ID NOS: 5-7, or to compliment thereof. In a related aspect, the invention provides a method for diagnosing the presence of a Aspergillus fumigatus in a subject sample that includes the steps of obtaining a DNA fraction from the subject sample; and performing a PCR amplification of the DNA fraction using at least one PCR primer that includes at least about 15 contiguous bases from any one of SEQ. ID NOS: 5-7, or a compliment thereof.

[0039] The present invention further provides an isolated nucleic acid molecule encoding a Candida glabrata Hsp60. In some embodiments, the isolated nucleotide molecule is selected from the group consisting of: (a) an isolated nucleic acid molecule comprising the sequence of SEQ ID NO:8 (FIG. 21); (b) an isolated nucleic acid molecule comprising the sequence of SEQ ID NO:8 from nucleotides 258-1964; (c) an isolated nucleic acid molecule comprising the sequence of SEQ ID NO:9 (FIG. 23); (d) an isolated nucleic acid molecule comprising the sequence of SEQ ID NO: 10 (FIG. 25); (e) an isolated nucleic acid molecule complementary to any one of the nucleotides of SEQ ID NOS: 8 to 10 set forth in (a) through (d), respectively.

[0040] In another aspect, the present invention provides an isolated nucleic acid molecule which is a variant of, or is substantially similar to, the Candida Hsp60 nucleotide molecules described above. In further aspects the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that is identical to a segment of contiguous nucleotide bases comprising at least 25% of SEQ ID NOS: 8 to 10 or a complement thereof or an isolated nucleic acid molecule encoding Hsp60 comprising a nucleic acid sequence that encodes a polypeptide comprising any one of the polypeptides according to FIGS. 21, 23 or 25, or a variant Hsp60 that is at least 95% homologous to a polypeptide according to any one of FIGS. 21, 23 or 25.

[0041] In one embodiment, the present invention provides an isolated nucleic acid molecule according as described above, the molecule encoding a polypeptide that is able to be selectively bound by an antibody specific for a Candida glabrata Hsp60.

[0042] In still another aspect in one aspect the present invention provides an isolated nucleic acid molecule encoding at least 8 contiguous amino acids of a Candida glabrata Hsp60 polypeptide selected from amino acid residues according to FIG. 21, wherein the encoded Candida glabrata Hsp60 polypeptide is able to bind to a major histocompatibility complex.

[0043] In still further aspects the present invention provides an isolated Candida glabrata Hsp60 polypeptide.

[0044] In some embodiments, the isolated Hsp60 polypeptide comprises the amino acid sequence of FIG. 21, or variants thereof, preferably wherein the polypeptide is able to be selectively bound by an antibody specific for a Candida glabrata Hsp60. In further embodiments, the isolated Hsp60 polypeptide is fused to an additional polypeptide to create a fusion protein.

[0045] In still yet further aspects the present invention provides an isolated Hsp60 polypeptide comprising at least 8 contiguous amino acids selected from amino acid residues according to FIG. 21, wherein the Hsp60 polypeptide is capable of binding to a major histocompatibility complex and eliciting or enhancing an immune response to Candida glabrata in a human being.

[0046] In certain embodiments, the isolated Hsp60 polypeptide is derived from proteolytic cleavage or chemical synthesis, or is an expression product of a transformed host cell containing a nucleic acid molecule encoding the Hsp60 or portion thereof. In further certain embodiments, the isolated Hsp60 polypeptide comprises greater than 95% homology to the Hsp60 polypeptide of FIG. 21, and the isolated Hsp60 polypeptide is able to be selectively bound by an antibody specific for a Candida glabrata Hsp60.

[0047] In still yet another aspect the present invention provides an isolated polypeptide wherein the polypeptide is an expression product of a transformed host cell containing at least one of the aforementioned nucleic acid molecules derived from Candida.

[0048] In still yet further aspects the present invention provides vectors comprising at least one of the aforementioned nucleic acid molecules derived from Candida. In certain embodiments, the vector is an expression vector comprising a promoter in operative linkage with the isolated nucleic acid molecule encoding the Hsp60 or portion thereof, preferably further comprising a selectable or identifiable marker and/or wherein the promoter is a constitutive or an inducible promoter. The present invention also provides host cells containing such vectors. In certain embodiments, the host cell is selected from the group consisting of a bacterial cell, a mammalian cell, a yeast cell, a plant cell and an insect cell.

[0049] In still yet other aspects the present invention provides compositions comprising a Candida Hsp60 polypeptide in combination with a pharmaceutically acceptable carrier or diluent. In certain embodiments, the composition is suitable for systemic administration, oral administration, intranasal administration or parenteral administration.

[0050] In yet other aspects the present invention provides methods for eliciting or enhancing an immune response in a mammal against Candida, comprising administering to the mammal in an amount effective to elicit or enhance the response, a Candida Hsp60 polypeptide in combination with a pharmaceutically acceptable carrier or diluent; methods for eliciting or enhancing an immune response in a mammal to a polypeptide comprising administering to the mammal a fusion protein containing sequences of the polypeptide fused to the Candida Hsp60 polypeptide in combination with a pharmaceutically acceptable carrier or diluent; and methods for eliciting or enhancing an immune response in a mammal against a target antigen comprising administering to the mammal the target antigen joined to a Candida Hsp60 polypeptide in combination with a pharmaceutically acceptable carrier or diluent.

[0051] In still another aspect, this invention provides PCR primers and probes for detecting DNA encoding a Candida glabrata Hsp60 that includes at least about 15 contiguous bases from any one of SEQ. ID NOS: 8-10, or to compliment thereof. In a related aspect, the invention provides a method for diagnosing the presence of Candida glabrata in a subject sample that includes the steps of obtaining a DNA fraction from the subject sample; and performing a PCR amplification of the DNA fraction using at least one PCR primer that includes at least about 15 contiguous bases from any one of SEQ. ID NOS: 8-10, or a compliment thereof.

[0052] These and other aspects of the present invention will become evident upon reference to the present specification and the attached drawings. In addition, various references are set forth herein that describe in more detail certain procedures or compositions (e.g., plasmids, etc.); all such references are incorporated herein in their entirety by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIG. 1 illustrates the strategy employed to obtain the nucleotide sequence of an internal fragment of the Neisseria meningitidis Hsp70 gene.

[0054] FIG. 2 depicts the nucleotide and amino acid sequences (SEQ ID Nos: 11 and 12, respectively) of an internal fragment of the Neisseria meningitidis Hsp70 gene.

[0055] FIG. 3 illustrates the strategy used to obtain the nucleotide and amino acid sequences of the Neisseria meningitidis Hsp70 gene.

[0056] FIG. 4 depicts first nucleotide and amino acid sequences of Neisseria mentingitidis Hsp70 gene (SEQ ID NO:1 and SEQ ID NO:13, respectively).

[0057] FIG. 5 illustrates strategy employed to obtain second nucleic acid and amino acid sequences of the Neisseria meningitidis Hsp70 gene.

[0058] FIG. 6 depicts second nucleotide and amino acid sequences of Neisseria meningitidis Hsp70 gene (SEQ ID NO:3 and SEQ ID NO:15, respectively).

[0059] FIG. 7 illustrates the strategy used to obtain nucleic acid and amino acid sequences of the Neisseria meningitidis Hsp70 genes cloned into pET24A+and pET28A+.

[0060] FIG. 8 depicts the nucleotide and amino acid sequences of Neisseria meningitidis hHsp70 gene cloned into pET24A+(SEQ ID NO:3 and SEQ ID NO: 15, respectively).

[0061] FIG. 9 depicts the nucleotide and amino acid sequences of Neisseria meningitidis Hsp70 gene cloned into pET28A+(SEQ ID NO:4 and SEQ ID NO: 16, respectively).

[0062] FIG. 10 shows a stained SDS-PAGE gel illustrating expression of recombinant Neisseria meningitidis Hsp70.

[0063] FIG. 11 shows a stained SDS-PAGE gel illustrating purification of recombinant Neisseria meningitidis Hsp70.

[0064] FIG. 12 shows an EtBr-stained gel illustrating selective amplification of Neisseria meningitidis Hsp70 and Streptococcal Hsp70 gene sequences.

[0065] FIG. 13 illustrates the strategy employed to obtain second nucleic acid and amino acid sequences of the Aspergillus fumigatus Hsp60 gene.

[0066] FIG. 14 depicts the nucleotide and amino acid sequences of Aspergillus fumigatus Hsp60 gene (SEQ ID NO:5 and SEQ ID Nos:17, 18, and 19, respectively).

[0067] FIG. 15 shows the map of expression plasmid pETAF60.

[0068] FIG. 16 depicts the nucleotide and amino acid sequences of Aspergillus fumigatus Hsp60 gene in plasmid pETAF60 (SEQ ID NO:6 and SEQ ID NO:20, respectively).

[0069] FIG. 17 shows the map of expression plasmid pETAF60H.

[0070] FIG. 18 depicts the nucleotide and amino acid sequences of Aspergillus fumigatus Hsp60 gene in plasmid pETAF60H (SEQ ID NO:7 and SEQ ID NO:21, respectively).

[0071] FIG. 19 shows a stained SDS-PAGE gel illustrating expression of recombinant Aspergillus fumigatus Hsp60.

[0072] FIG. 20 illustrates the strategy employed to obtain nucleic acid and amino acid sequences of the Candida glabrata Hsp60 gene.

[0073] FIG. 21 depicts the nucleotide and amino acid sequences of Candida glabrata Hsp60 gene (SEQ ID NO:8 and SEQ ID NO:22, respectively).

[0074] FIG. 22 shows the map of expression plasmid pETCG60A.

[0075] FIG. 23 depicts the nucleotide and amino acid sequences of Candida glabrata Hsp60 gene in plasmid pETCG60A (SEQ ID NO:9 and SEQ ID NO:23, respectively).

[0076] FIG. 24 shows the map of expression plasmid pETCG60AH.

[0077] FIG. 25 depicts the nucleotide and amino acid sequences of Candida glabrata Hsp60 gene in plasmid pETCGA60H (SEQ ID NO: 10 and SEQ ID NO:24, respectively).

[0078] FIG. 26 shows a stained SDS-PAGE gel illustrating expression of recombinant Candida glabrata Hsp60.

[0079] FIG. 27 shows a stained SDS-PAGE gel illustrating purification of recombinant Candida glabrata Hsp60.

DETAILED DESCRIPTION OF THE INVENTION

[0080] The present invention provides methods and compositions comprising isolated nucleic acid molecules and polypeptides specific to Neisseria meningitidis, Aspergillus fumigatus and Candida glabrata, as well as vector constructs, antibodies and other materials related to isolated nucleic acid molecules and polypeptides. Such compositions and methods are useful for the diagnosis of Neisserial, Aspergillal and Candidal infection and for generating (eliciting or enhancing) an immune response to these organisms.

[0081] Prior to setting forth the invention, it may be helpful to an understanding thereof to set forth definitions of certain terms to be used hereafter.

[0082] A "stress gene," also known as "heat shock gene," is a gene that is activated or otherwise detectably upregulated due to the contact or exposure of an organism (containing the gene) to a stressor, such as heat shock or glucose deprivation or glucose addition. A given "stress gene" also includes homologous genes within known stress gene families, such as certain genes within the Hsp60, Hsp70 and Hsp90 stress gene families, even though such homologous genes are not themselves induced by a stressor.

[0083] A "stress protein," also known as a "heat shock protein," ("Hsp") is a protein that is encoded by a stress gene, and is therefore typically produced in significantly greater amounts upon the contact or exposure to the stressor of the organism. Each of the terms stress gene and stress protein as used in the present specification are inclusive of the other, unless the context indicates otherwise. Neisserial, Aspergillal and Candidal Hsps, as well as Hsps from other organisms, appear to participate in important cellular processes such as protein synthesis and assembly and disassembly of protein complexes.

[0084] As used herein, "polypeptide" refers to full length proteins and fragments thereof.

[0085] As used herein, "peptide" refers to a fragment of the whole protein, whether chemically or biologically produced.

[0086] As used herein, "immunogenic" refers to an antigen or composition that elicits an immune response.

[0087] An "isolated nucleic acid molecule" refers to a polynucleotide molecule, in the form of a separate fragment or as a component of a larger nucleic acid construct, that has been separated from its source cell (including the chromosome it normally resides in) at least once in a substantially pure form. Nucleic acid molecules can be comprised of a wide variety of nucleotides and molecules well known in the art, including DNA, RNA, nucleic acid analogues, or any combination of these.

[0088] As used herein, "vector" refers to a polynucleotide assembly capable of directing expression and/or replication of the nucleic acid sequence of interest. Such assembly can, if desired, be included as a part of other components, such as a protein, lipid or lipoprotein coat, for delivery of the vector or for other purposes.

[0089] An "expression vector" refers to polynucleotide vector having at least a promoter sequence operably linked to the nucleic acid sequence of interest.

[0090] As used herein, a "promoter" refers to a nucleotide sequence that contains elements that direct the transcription of an operably linked nucleic acid sequence. At minimum, a promoter contains an RNA polymerase binding site. Promoter regions can also contain enhancer elements which by definition enhance transcription.

A. Hsp Genes Anf Polypeptides from Neisseria Meningitidis, Aspergillus Fumigatus and Candida Glabrata

[0091] As used herein, "Hsp70" refers to heat shock genes from a Hsp70 family of genes that encode heat shock proteins of approximately 70 kDa, and the heat shock gene products encoded thereby. The nucleotide and amino acid sequences of Hsp70 genes and gene products from Neisseria meningitidis are set forth in FIGS. 4, 6, 8 and 9 SEQ ID NOS: 1, 3 and 4 (nucleotide) and SEQ ID NOS: 13, 15 and 16 (amino acid); such sequences also include the PCR primers used to isolate the Hsp70 genes). As used herein, Hsp60 refers to heat shock genes from the Hsp60 family of genes that encode heat shock proteins of approximately 60 kDa; and the heat shock gene products encoded thereby. The nucleotide and amino acid sequences of Hsp60 genes and gene products from Aspergillus fumigatus and Candida glabrata are set forth in FIGS. 14, 16, 18, 21, 23 and 25 (SEQ ID NOS: 5-10; such sequences also include the PCR primers used to isolate the Hsp60 genes).

[0092] Within the context of this invention, it should be understood that Hsp70 and Hsp60 include wild-type/native protein sequences, as well as other variants (including alleles) and fragments of the native protein sequences. Briefly, such variants may result from natural polymorphisms or be synthesized by recombinant methodology or chemical synthesis, and differ from wild-type proteins by one or more amino acid substitutions, insertions, deletions, or the like. Further, in the region of homology to the native sequence, a variant should preferably have at least 95% amino acid sequence homology, and within certain embodiments, greater than 97% or 98% homology. As used herein, amino acid "homology" is determined by a computer algorithm incorporated in a protein database search program commonly used in the art, and more particularly, as incorporated in the programs BLAST.TM. (Altschul et al., Nucleic Acids Res. (25) 3389-3402, 1997) or DNA STAR MEGALIGN.TM. which return similar results in homology calculations. As will be appreciated by those of ordinary skill in the art, a nucleotide sequence encoding an Hsp or a variant may differ from the native sequences presented herein due to codon degeneracies, nucleotide polymorphisms, or nucleotide substitutions, deletions or insertions.

[0093] The aforementioned sequences are useful for generating PCR primers and probes for the detection of Neisseria meningitidis, Aspergillus fumigatus or Candida glabrata. Thus, one aspect of this invention includes PCR primers and probes for detecting DNA encoding the Hsps disclosed herein. Useful PCR primers typically include at least about 15 contiguous bases from the nucleic acid sequences provided herein, or compliments thereof. More particularly, PCR primers and probes for detection of Neisseria meningitidis include at least about 15 contiguous bases from any one of SEQ ID NOS: 1, 3 and 4 or compliments thereof; PCR primers and probes for detection of Aspergillus fumigatus include at least about 15 contiguous bases from any one of SEQ. ID NOS: 5-7 or compliments thereof; and PCR primers and probes for detection of Candida glabrata include at least about 15 contiguous bases from any one of SEQ. ID NOS: 8-10 or compliments thereof. In certain embodiments, PCR primers derived from these sequences can be used in a diagnostic method to detect the presence of a specific microorganism in a subject sample by amplifying DNA isolated from the subject sample to detect the Hsp genes present in any one of Neisseria meningitidis, Aspergillus fumigatus or Candida glabrata as opposed to other microorganisms. Thus for example, primer pairs comprising 5' CTGCCGTACATCACCATGG 3' (SEQ ID NO:25) with 5' GGCTTCTTGTACTTTCGGC -3' (SEQ ID NO:26); were able to specifically amplify DNA isolated from Neisseria meningitidis, but not other microorganisms known to contain Hsps. Similarly, primer pairs derived from an Hsp70 gene isolated from Streptococcus (5' TGACCTTGTTGAACGTAC -3' (SEQ ID:27) with 5'ACTTCATCAGGGTTTAC -3' (SEQ ID NO:28)) were able to amplify DNA isolated from Streptococcus but not Neisseria (See Example 5).

[0094] An "isolated nucleic acid molecule encoding Neisseria meningitidis Hsp70, Aspergillus fumigatus Hsp60 or Candida glabrata Hsp60" refers to nucleic acid sequences that are capable of encoding Hsp70 or Hsp60 polypeptides of these organisms. While several embodiments of such molecules are depicted in SEQ ID NOS: 1 and 3-10, it should be understood that within the context of the present invention, reference to one or more of these molecules includes variants that are naturally occurring and/or synthetic sequences which are substantially similar to the sequences provided herein and, where appropriate, the protein (including peptides and polypeptides) that are encoded by these sequences and their variants. As used herein, the nucleotide sequence is deemed to be "substantially similar" if: (a) the nucleotide sequence is derived from the coding region of a native gene of Neisseria meningitidis, Aspergillus fumigatus or Candida glabrata and encodes a peptide or polypeptide that binds to an antibody or HLA molecule that specifically binds to a polypeptide described herein (including, for example, portions of the sequence or allelic variations of the sequences discussed above); or (b) the nucleotide sequences are degenerate (i.e., sequences which encode the same amino acid using a different codon sequence) as a result of the genetic code to the nucleotide sequences defined in (a); or (c) the nucleotide sequence is at least 95% identical to a nucleotide sequence provide herein, or (d) is a complement of any of the sequences described in (a-c). As used herein, "high stringency" are conditions for hybridization of nucleic acids as described in units 6.3 and 6.4 by Ausubel et al., Current Protocols in Molecular Biology, vol. 1. John Wiley & Sons (1998).

[0095] One aspect of the present invention is the use of Neisseria meningitidis Hsp70, Aspergillus fumigatus Hsp60 or Canadida glabrata Hsp60 nucleotide sequences to produce recombinant proteins for immunizing an animal. Therefore, the use of any length of nucleic acid disclosed by the present invention (preferably 24 nucleotides or longer) which encodes a polypeptide or fragment thereof that is capable of binding to the major histocompatibility complex and eliciting or enhancing an immunogenic response is contemplated by this invention. Immunogenic response can be readily tested by known methods such as challenging a mouse or rabbit with the antigen of interest and thereafter collecting plasma and determining if antibodies of interest are present. Other assays particularly useful for the detection of T-cell responses include proliferation assays, T-cell cytotoxicity assays and assays for delayed hypersensitivity. In determining whether an antibody specific for the antigen of interest was produced by the animal, many diagnostic tools are available, for example, testing binding of labeled antigen to plasma derived antibodies, or using Enzyme-linked immunoassays with tag attached to the antigen of interest.

[0096] The Neisseria meningitidis Hsp70, Aspergillus fumigatus Hsp60 and Canadida glabrata Hsp60 genes of this invention can be obtained using a variety of methods. For example, a nucleic acid molecule can be obtained from a cDNA or genomic expression library by screening with an antibody or antibodies reactive to one or more of these Hsps (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1989; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987). Further, random-primed PCR can be employed (see, e.g., Methods in Enzymol. 254:275, 1995). In one such method, one of the primers is a poly deoxy-thymine and the other is a degenerate primer based on the amino acid sequence or nucleotide sequence of related Hsps.

[0097] Other methods can also be used to obtain a nucleic acid molecule that encodes Neisseria meningitidis Hsp70, Aspergillus fumigatus Hsp60 or Canadida glabrata Hsp60. For example, a nucleic acid molecule can be obtained by using the sequence information provided herein to synthesize a probe which can be labeled, such as with a radioactive label, enzymatic label, protein label, fluorescent label, or the like, and hybridized to a genomic library or a cDNA library constructed in a phage, plasmid, phagemid, viral, or other vector (see, e.g., Sambrook et al. (supra); Ausubel et al. (supra)). DNA representing RNA or genomic nucleic acid sequence can also be obtained by amplification using sets of primers complementary to 5' and 3' sequences of the cDNA sequence, such as presented in the Examples. For ease of cloning, restriction sites can also be incorporated into the primers.

[0098] Variants (including alleles) of the Hsp70 and Hsp60 genes provided herein can be readily isolated from natural sources containing such variants (e.g., polymorphisms, mutants), or can be synthesized or constructed using recombinant DNA and mutagenesis techniques known in the art. Many methods have been developed for generating mutants (see generally Sambrook et al. (supra); Ausubel et al. (supra)). Briefly, preferred methods for generating nucleotide substitutions utilize an oligonucleotide that spans the base or bases to be mutated and contains the mutated base or bases. The oligonucleotide is hybridized to complementary single stranded nucleic acid and second strand synthesis is primed from the oligonucleotide. The double-stranded nucleic acid is prepared for transformation into host cells, such as E. coli, other prokaryotes, yeast or other eukaryotes. Standard screening and vector growth protocols are used to identify mutant sequences and obtain high yields.

[0099] Similarly, deletions and/or insertions of the Hsp70 or the Hsp60 gene can be constructed by any of a variety of known methods. For example, the gene can be digested with restriction enzymes and religated such that sequence is deleted or religated with additional sequence such that an insertion or large substitution is made. Other means of generating variant sequences, known in the art, can be employed, for examples see Sambrook et al. (supra) and Ausubel et al. (supra). Moreover, verification of variant sequences is typically accomplished by restriction enzyme mapping, sequence analysis or hybridization. Variants which encode a polypeptide that elicits an immunogenic response specific for Neisseria meningitidis, Aspergillus fumigatus or Candida glabrata are useful in the context of this invention.

[0100] As noted above, the present invention also provides isolated polypeptides. Within the context of the present invention, unless otherwise clear from the context, such polypeptides are understood to include the whole, or portions/fragments, of a gene product derived from one or more of the Hsp70 and Hsp60 genes of the invention or variants thereof as discussed above. In one aspect of the present invention, the protein is encoded by a portion of a native gene or is encoded by a variant of a native gene and the protein or fragment thereof elicits or enhances an immune response specific for Neisseria meningitidis, Aspergillus fumigatus or Candida glabrata.

[0101] A "purified" Hsp70 or Hsp60 stress protein of the present invention is a heat shock protein of the Hsp70 or Hsp60 family from Neisseria meningitidis (Hsp70), Aspergillus fumigatus (Hsp60 ) or Candida glabrata (Hsp60 ) that has been purified from its producing cell. For example, the Hsp70 and Hsp60 polypeptides of the present invention can be purified by a variety of standard methods with or without a detergent purification step. For example, Hsp70 or Hsp60 of the present invention can be isolated by, among other methods, culturing suitable host and vector systems to produce recombinant Hsps (discussed further herein). Then, supernatants from such cell lines, or Hsp inclusions, or whole cells where the Hsp is not excreted into the supernatant, can be treated by a variety of purification procedures. For example, the Hsp-containing composition can be applied to a suitable purification matrix such as an anti-Hsp60 antibody bound to a suitable support. Alternatively, anion or cation exchange resins, gel filtration or affinity, hydrophobic or reverse phase chromatography may be employed in order to purify the protein. The Hsp polypeptide can also be concentrated using commercially available protein concentration filters, such as an Amicon or Millipore Pellicon ultrafiltration unit, or by vacuum dialysis. In another alternative, when the polypeptide is secreted the supernatant medium containing the polypeptide can first be concentrated using one of the above mentioned protein concentration filters, followed by application of the concentrate to a suitable purification matrix such as those described above.

[0102] In one embodiment, the isolated Hsp70 and Hsp60s of the present invention are produced in a recombinant form, utilizing genetic manipulation techniques that are well known in the art. For example, a Hsp of the present invention can be expressed as a histidine-tagged molecule, permitting purification on a nickel-chelating matrix. Alternatively, other tags may be used, including FLAG and GST. The associated tag can then be removed in the last step of purification, for example, for certain vectors, His-tagged proteins may be incubated with thrombin, resulting in cleavage of a recognition sequence between the tag and the Hsp polypeptide (e.g., pET vectors from Invitrogen). Following purification of an Hsp of the invention from a gram-negative bacterial host, whether tagged or not, it will be necessary to reduce the level of endotoxin in the Hsp preparation.

B. Vectors, Host Cells, and Expression of Hsps from Neisseria Meningitidis, Aspergillus Fumigatus and Candida Glabrata

[0103] It is well known in the art that certain vectors (e.g., pUC) can be used for producing multiple copies of a nucleotide molecule of interest as well as being useful for genetic manipulation techniques (e.g., site-directed mutagenesis). See Sambrook (supra). Expression vectors are particularly suited to the practice of this invention. An expression vector includes transcriptional promoter/enhancer elements operably linked to the Neisserial, Aspergillal or Candidal Hsp nucleic acid molecule and may typically contain a selectable or otherwise identifiable marker gene. The expression vector may be composed of either deoxyribonucleic acids ("DNA"), ribonucleic acids ("RNA"), or a combination of the two (e.g., a DNA-RNA chimera). Optionally, the expression vector may include a polyadenylation sequence or one or more restriction sites. Additionally, depending on the host cell chosen and the expression vector employed, other genetic elements such as an origin of replication, additional nucleic acid restriction sites, enhancers, sequences conferring inducibility of transcription, and genes encoding proteins suitable for use as selectable or identifiable markers, may also be incorporated into the expression vectors described herein.

[0104] The manipulation and expression of Neisserial, Aspergillal or Candidal Hsp genes can be accomplished by culturing host cells containing an expression vector capable of expressing the Hsp genes. Such vectors or vector constructs include either synthetic or cDNA-derived nucleic acid molecules or genomic DNA fragments encoding the Hsp polypeptides, which are operably linked to suitable transcriptional or translational regulatory elements. Suitable regulatory elements within the expression vector can be derived from a variety of sources, including bacterial, fungal, viral, mammalian, insect, or plant genes. Selection of appropriate regulatory elements is dependent on the host cell chosen, and can be readily accomplished by one of ordinary skill in the art in light of the present specification. Examples of regulatory elements include a transcriptional promoter and enhancer or RNA polymerase binding sequence, a transcriptional terminator, and a ribosomal binding sequence, including a translation initiation signal.

[0105] Nucleic acid molecules that encode any of the Neisserial, Aspergillal or Candidal Hsp polypeptides described above can be expressed by a wide variety of prokaryotic and eukaryotic host cells, including bacterial, mammalian, yeast or other fungi, viral, insect, and plant cells. The selection of a host cell may also assist the production of post-translationally modified Hsps (e.g. modified by glycosylation, prenylation, acetylation or other processing event), depending upon the desires of the user. Methods for transforming or transfecting such cells to express nucleic acids are well known in the art (see, e.g., Itakura et al., U.S. Pat. No. 4,704,362; Hinnen et al., PNAS USA 75:1929-1933, 1978; Murray et al., U.S. Pat. No. 4,801,542; Upshall et al., U.S. Pat. No. 4,935,349; Hagen et al., U.S. Pat. No. 4,784,950; Axel et al., U.S. Pat. No. 4,399,216; Goeddel et al., U.S. Pat. No. 4,766,075; and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2.sup.nd edition, Cold Spring Harbor Laboratory Press, 1989; for plant cells see Czako and Marton, Plant Physiol. 104:1067-1071, 1994; Paszkowski et al., Biotech. 24:387-392, 1992).

[0106] Bacterial host cells suitable for carrying out the present invention include E. coli, such as E. coli DH5a (Stratagene, La Jolla, Calif.) or BL21 (DE3) (Novagen, Madison, Wis.), M. leprae, M tuberculosis, M. bovis, B. subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, Streptococcus, and Staphylococcus, as well as many other bacterial species well known to one of ordinary skill in the art.

[0107] Bacterial expression vectors preferably comprise a promoter, which functions in the host cell, one or more selectable phenotypic markers, and a bacterial origin of replication. Representative promoters include the .beta.-lactamase (penicillinase) and lactose promoter system (see Chang et al., Nature 275:615, 1978), the T7 RNA polymerase promoter (Studier et al., Meth. Enzymol. 185:60-89, 1990), the lambda promoter (Elvin et al., Gene 87:123-126, 1990), the trp promoter (Nichols and Yanofsky, Meth. in Enzymology 101: 155, 1983) and the tac promoter (Russell et al., Gene 20: 231, 1982). Representative selectable markers include various antibiotic resistance markers such as the kanamycin or ampicillin resistance genes. Many plasmids suitable for transforming host cells are well known in the art, including among others, pBR322 (see Bolivar et al., Gene 2:95, 1977), the pUC plasmids pUC 18, pUC19, pUC118, pUC119 (see Messing, Meth. in Enzymology 101:20-77, 1983; Vieira and Messing, Gene 19:259-268, 1982), and pNH8A, pNH16a, pNH18a, and Bluescript M13 (Stratagene, La Jolla, Calif).

[0108] Fungal host cells suitable for carrying out the present invention include, among others, Saccharomyces pombe, Saccharomyces cerevisiae, the genera Pichia or Kluyveromyces and various species of the genus Aspergillus (McKnight et al., U.S. Pat. No. 4,935,349). Suitable expression vectors for yeast and fungi include, among others, YCp50 (ATCC No. 37419) for yeast, and the amdS cloning vector pV3 (Turnbull, Bio/Technology 7:169, 1989), YRp7 (Struhl et al., Proc. Natl. Acad. Sci. USA 76:1035-1039, 1978), YEp13 (Broach et al., Gene 8:121-133, 1979), pJDB249 and pJDB219 (Beggs, Nature 275:104-108, 1978) and derivatives thereof.

[0109] Preferred promoters for use in yeast include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255:12073-12080, 1980; Alber and Kawasaki, J. Mol. Appl. Genet. 1:419-434, 1982) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals, Hollaender et al. (eds.), p. 355, Plenum, N.Y., 1982; Ammerer, Meth. Enzymol. 101:192-201, 1983). Examples of useful promoters for fungi vectors include those derived from Aspergillus nidulans glycolytic genes, such as the adh3 promoter (McKnight et al., EMBO J 4:2093-2099, 1985). The expression units may also include a transcriptional terminator. An example of a suitable terminator is the adh3 terminator (McKnight et al., ibid., 1985).

[0110] As with bacterial vectors, the yeast vectors will generally include a selectable marker, which may be one of any number of genes that exhibit a dominant phenotype for which a phenotypic assay exists to enable transformants to be selected. Preferred selectable markers include those that complement host cell auxotrophy, provide antibiotic resistance or enable a cell to utilize specific carbon sources, and include leu2 (Broach et al., ibid.), ura3 (Botstein et al., Gene 8:17, 1979), or his3 (Struhl et al., ibid.). Another suitable selectable marker is the cat gene, which confers chloramphenicol resistance on yeast cells.

[0111] Techniques for transforming fungi are well known in the literature, and have been described, for instance, by Beggs (ibid.), Hinnen et al.(Proc. Natl. Acad. Sci. USA 75:1929-1933, 1978), Yelton et al. (Proc. Natl. Acad. Sci. USA 81:1740-1747, 1984), and Russell (Nature 301:167-169, 1983). The genotype of the host cell may contain a genetic defect that is complemented by the selectable marker present on the expression vector. Choice of a particular host and selectable marker is well within the level of ordinary skill in the art in light of the present specification.

[0112] Protocols for the transformation of yeast are also well known to those of ordinary skill in the art. For example, transformation may be readily accomplished either by preparation of spheroplasts of yeast with DNA (see Hinnen et al., PNAS USA 75:1929, 1978) or by treatment with alkaline salts such as LiCl (see Itoh et al., J. Bacteriology 153:163, 1983). Transformation of fingi may also be carried out using polyethylene glycol as described by Cullen et al. (Bio/Technology 5:369, 1987).

[0113] Viral vectors include expression vectors that comprise a promoter which directs the expression of an isolated nucleic acid molecule encoding a Hsp according to this invention. A wide variety of promoters may be utilized within the context of the present invention, including for example, promoters such as MoMLV LTR, RSV LTR, Friend MuLV LTR, adenoviral promoter (Ohno et al., Science 265: 781-784, 1994), neomycin phosphotransferase promoter/enhancer, late parvovirus promoter (Koering et al., Hum. Gene Therap. 5:457-463, 1994), Herpes TK promoter, SV40 promoter, metallothionein Ia gene enhancer/promoter, cytomegalovirus immediate early promoter, and the cytomegalovirus immediate late promoter. The promoter may also be a tissue-specific promoter (see e.g., WO 91/02805; EP 0,415,731; and WO 90/07936). In addition to the above-noted promoters, other viral-specific promoters (e.g., retroviral promoters (including those noted above, as well as others such as HIV promoters), hepatitis, herpes (e.g., EBV), and bacterial, fungal or parasitic-specific (e.g., malarial-specific) promoters may be utilized in order to target a specific cell or tissue which is infected with a virus, bacteria, fungus or parasite.

[0114] Thus, Neisserial Hsp70, Aspergillal Hsp60 or Candidal Hsp60 polypeptides of the present invention may be expressed from a variety of viral vectors, including for example, herpes viral vectors (e.g., U.S. Pat. No. 5,288,641), adenoviral vectors (e.g., WO 94/26914, WO 93/9191; Kolls et al., PNAS 91(1):215-219, 1994; Kass-Eisler et al., PNAS 90(24):11498-502, 1993; Guzman et al., Circulation 88(6):2838-48, 1993; Guzman et al., Cir. Res. 73(6):1202-1207, 1993; Zabner et al., Cell 75(2):207-216, 1993; Li et al., Hum Gene Ther. 4(4):403-409, 1993; Caillaud et al., Eur. J. Neurosci. 5(10):1287-1291, 1993; Vincent et al., Nat. Genet. 5(2):130-134, 1993; Jaffe et al., Nat. Genet. 1(5):372-378, 1992; and Levrero et al., Gene 101(2):195-202, 1991), adenovirus-associated viral vectors (Flotte et al., PNAS 90(22): 10613-10617, 1993), baculovirus vectors, parvovirus vectors (Koering et al., Hum. Gene Therap. 5:457-463, 1994), pox virus vectors (Panicali and Paoletti, PNAS 79:4927-4931, 1982; and Ozaki et al., Biochem. Biophys. Res. Comm. 193(2):653-660, 1993), and retroviruses (e.g., EP 0,415,731; WO 90/07936; WO 91/0285, WO 94/03622; WO 93/25698; WO 93/25234; U.S. Pat. No. 5,219,740; WO 93/11230; WO 93/10218. Within various embodiments, either the viral vector itself or a viral particle which contains the viral vector may be utilized in the methods and compositions described below.

[0115] Mammalian cells suitable for carrying out the present invention include, among others: PC12 (ATCC No. CRL1721), NIE-115 neuroblastoma, SK-N-BE(2)C neuroblastoma, SHSY5 adrenergic neuroblastoma, NS20Y and NG108-15 murine cholinergic cell lines, or rat F2 dorsal root ganglion line, COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g., ATCC No. CRL 6281; BHK 570 cell line (deposited with the American Type Culture Collection under accession number CRL 10314), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCC No. 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and NS-1 cells. Other mammalian cell lines may be used within the present invention, including Rat Hep I (ATCC No. CRL 1600), Rat Hep II (ATCC No. CRL 1548), TCMK (ATCC No. CCL 139), Human lung (ATCC No. CCL 75.1), Human hepatoma (ATCC No. HTB-52), Hep G2 (ATCC No. HB 8065), Mouse liver (ATCC No. CCL 29.1), NCTC 1469 (ATCC No. CCL 9.1), SP2/0-Agl4 (ATCC No. 1581), HIT-T1 5 (ATCC No. CRL 1777), and RINm 5AHT2B (Orskov and Nielson, FEBS 229(1): 175-178, 1988).

[0116] Mammalian expression vectors for use in carrying out the present invention include a promoter capable of directing the transcription of a cloned gene or cDNA. Preferred promoters include viral promoters and cellular promoters. Example viral promoters include the cytomegalovirus immediate early promoter (Boshart et al., Cell 41:521-530, 1985), cytomegalovirus immediate late promoter, SV40 promoter (Subramani et al., Mol. Cell. Biol. 1:854-864, 1981), MMTV LTR, RSV LTR, and adenovirus E1a. Example cellular promoters include the mouse metallothionein-1 promoter (Palmiter et al., U.S. Pat. No. 4,579,821), actin promoters, a mouse VH promoter (Bergman et al., Proc. Natl. Acad Sci. USA 81:7041-7045, 1983; Grant et al., Nuc. Acids Res. 15:5496, 1987) and a mouse V.sub.H promoter (Loh et al., Cell 33:85-93, 1983). The choice of promoter will depend, at least in part, upon the level of expression desired or the recipient cell line to be transfected.

[0117] Such expression vectors can also contain a set of RNA splice sites located downstream from the promoter and upstream from the DNA sequence encoding the peptide or protein of interest. Preferred RNA splice sites may be obtained from adenovirus and/or immunoglobulin genes. Also contained in the expression vectors is a polyadenylation signal located downstream of the coding sequence of interest. Suitable polyadenylation signals include the early or late polyadenylation signals from SV40 (Kaufman and Sharp, ibid.), the polyadenylation signal from the Adenovirus 5 E1B region and the human growth hormone gene terminator (DeNoto et al., Nuc. Acids Res. 9:3719-3730, 1981). The expression vectors may include a noncoding viral leader sequence, such as the Adenovirus 2 tripartite leader, located between the promoter and the RNA splice sites. Preferred vectors may also include enhancer sequences, such as the SV40 enhancer. Expression vectors may also include sequences encoding the adenovirus VA RNAs. Suitable expression vectors can be obtained from commercial sources (e.g., Stratagene, La Jolla, Calif.).

[0118] Vector constructs comprising cloned DNA sequences can be introduced into cultured mammalian cells by, for example, calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-845, 1982), or DEAE-dextran mediated transfection (Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987). See generally Sambrook et al. (supra). To identify cells that have stably integrated the cloned DNA, a selectable marker is generally introduced into the cells along with the gene or cDNA of interest. Preferred selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate. The selectable marker may be an amplifiable selectable marker. Preferred amplifiable selectable markers are the DHFR gene and the neomycin resistance gene. Selectable markers are reviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass.).

[0119] Mammalian cells containing a suitable vector are allowed to grow for a period of time, typically 1-2 days, to begin expressing the DNA sequence(s) of interest. Drug selection is then applied to select for growth of cells that are expressing the selectable marker in a stable fashion. For cells that have been transfected with an amplifiable, selectable marker the drug concentration may be increased in a stepwise manner to select for increased copy number of the cloned sequences, thereby increasing expression levels. Cells expressing the introduced sequences are selected and screened for production of the protein of interest in the desired form or at the desired level. Cells that satisfy these criteria can then be cloned and scaled up for production.

[0120] Numerous insect host cells known in the art can also be useful within the present invention, in light of the subject specification. For example, the use of baculoviruses as vectors for expressing heterologous DNA sequences in insect cells has been reviewed by Atkinson et al. (Pestic. Sci. 28:215-224,1990).

[0121] Numerous plant host cells known in the art can also be useful within the present invention, in light of the subject specification. For example, the use of Agrobacterium rhizogenes as vectors for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987.

[0122] Upon expression of the Neisserial Hsp70, Aspergillal Hsp60 or Candidal Hsp60 polypeptides or fragments thereof in the host cells, the polypeptide or peptide may be released and/or isolated from the host cell utilizing methods such as those discussed previously herein.

[0123] As noted above, depending on the host cell in which one desires to express an Neisserial, Aspergillal or Candidal Hsp, the gene encoding the protein is introduced into an expression vector comprising a promoter that is active in the host cell. Other components of the expression unit such as transcribed but not translated sequences at the ends of the coding region may also be selected according to the particular host utilized. In some cases, it may be necessary to introduce artificially an intervening sequence to ensure high level expression. Expression can be monitored by SDS-PAGE and staining, if expression levels are sufficiently high. Additionally, if the protein is produced with a tag, detection by anti-tag antibody can be carried out and if produced with no tag, detection by anti-Hsp antibody that does not recognize homologous proteins of the host may be employed. Further, any method known in the art for protein identification may be utilized to this end (e.g., a high resolution electrophoretic method or 2D electrophoresis).

C. Preparation of Antibodies Against the Hsp Polypeptides of the Present Invention

[0124] In another aspect, the proteins of the present invention are utilized to prepare specifically binding antibodies (i.e., binding partners). Accordingly, the present invention also provides such antibodies. Within the context of the present invention, the term "antibodies" includes polyclonal antibodies, monoclonal antibodies, anti-idiotypic antibodies, fragments thereof such as F(ab').sub.2 and Fab fragments, and recombinantly or synthetically produced binding partners. Such binding partners incorporate the variable regions that permit an antibody to specifically bind, which means an antibody able to selectively bind to a peptide produced from one of the Neisserial, Aspergillal or Candidal Hsp genes of the invention with a Kd of about 10.sup.-3 M or less. The affinity of an antibody or binding partner can be readily determined by one of ordinary skill in the art (see Scatchard, Ann. N. Y Acad. Sci. 51:660-672, 1949).

[0125] Polyclonal antibodies can be readily generated by one of ordinary skill in the art from a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, turkeys, rabbits, mice, or rats. Briefly, the desired protein or peptide is utilized to immunize the animal through intraperitoneal, intramuscular, intraocular, or subcutaneous injections. The immunogenicity of the protein or peptide of interest may be increased through the use of an adjuvant such as Freund's complete or incomplete adjuvant. Following several booster immunizations, small samples of serum are collected and tested for reactivity to the desired protein or peptide.

[0126] Typically, suitable polyclonal antisera give a signal that is at least three times greater than background. Once the titer of the animal has reached a plateau in terms of its reactivity to the protein, larger quantities of polyclonal antisera may be readily obtained either by weekly bleedings, or by exsanguinating the animal.

[0127] Monoclonal antibodies can also be readily generated using well-known techniques (see U.S. Pat. Nos. 32,011, 4,902,614, 4,543,439, and 4,411,993; see also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKeam, and Bechtol (eds.), 1980, and Antibodies. A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988). Briefly, in one embodiment, a subject animal such as a rat or mouse is injected with a desired protein or peptide. If desired, various techniques may be utilized in order to increase the resultant immune response generated by the protein, in order to develop greater antibody reactivity. For example, the desired protein or peptide may be coupled to another protein such as ovalbumin or keyhole limpet hemocyanin (KLH), or through the use of adjuvants such as Freund's complete or incomplete adjuvant. The initial elicitation of an immune response, may preferably be through intraperitoneal, intramuscular, intraocular, or subcutaneous routes.

[0128] Between one and three weeks after the initial immunization, the animal may be reimmunized. The animal may then be test bled and the serum tested for binding to the desired antigen using assays as described above. Additional immunizations may also be accomplished until the animal has reached a plateau in its reactivity to the desired protein or peptide. The animal may then be given a final boost of the desired protein or peptide, and three to four days later sacrificed. At this time, the spleen and lymph nodes may be harvested and disrupted into a single cell suspension by passing the organs through a mesh screen or by rupturing the spleen or lymph node membranes which encapsulate the cells. Within one embodiment the red cells are subsequently lysed by the addition of a hypotonic solution, followed by immediate return to isotonicity.

[0129] Within another embodiment, suitable cells for preparing monoclonal antibodies are obtained through the use of in vitro immunization techniques. Briefly, an animal is sacrificed, and the spleen and lymph node cells are removed as described above. A single cell suspension is prepared, and the cells are placed into a culture containing a form of the protein or peptide of interest that is suitable for generating an immune response as described above. Subsequently, the lymphocytes are harvested and fused as described below.

[0130] Cells that are obtained through the use of in vitro immunization or from an immunized animal as described above may be immortalized by transfection with a virus such as the Epstein-Barr Virus (EBV). (See Glasky and Reading, Hybridoma 8(4):377-389, 1989.) Alternatively, within a preferred embodiment, the harvested spleen and/or lymph node cell suspensions are fused with a suitable myeloma cell in order to create a "hybridoma" which secretes monoclonal antibodies. Suitable myeloma lines are preferably defective in the construction or expression of antibodies, and are additionally syngeneic with the cells from the immunized animal. Many such myeloma cell lines are well known in the art and may be obtained from sources such as the American Type Culture Collection (ATCC), Rockville, Md. (see Catalogue of Cell Lines & Hybridomas, 6.sup.th ed., ATCC, 1988). Representative myeloma lines include: for humans, UC 729-6 (ATCC No. CRL 8061), MC/CAR-Z2 (ATCC No. CRL 8147), and SKO-007 (ATCC No. CRL 8033); for mice, SP2/0-Ag41 (ATCC No. CRL 1581), and P3X63Ag8 (ATCC No. TIB 9); and for rats, Y3-Ag1.2.3 (ATCC No. CRL 1631), and YB2/0 (ATCC No. CRL 1662). Particularly preferred fusion lines include NS-1 (ATCC No. TIB 18) and P3X63--Ag 8.653 (ATCC No. CRL 1580), which may be utilized for fusions with either mouse, rat, or human cell lines. Fusion between the myeloma cell line and the cells from the immunized animal can be accomplished by a variety of methods, including the use of polyethylene glycol (PEG) (see Antibodies: A Laboratory Manual, Harlow and Lane, supra) or electrofusion. (See Zimmerman and Vienken, J. Membrane Biol. 67:165-182, 1982.)

[0131] Following the fusion, the cells are placed into culture plates containing a suitable medium, such as RPMI 1640 or DMEM (Dulbecco's Modified Eagles Medium, JRH Biosciences, Lenexa, Kans.). The medium may also contain additional ingredients, such as Fetal Bovine Serum (FBS, e.g., from Hyclone, Logan, Utah, or JRH Biosciences), thymocytes that were harvested from a baby animal of the same species as was used for immunization, or agar to solidify the medium. Additionally, the medium should contain a reagent which selectively allows for the growth of fused spleen and myeloma cells. Particularly preferred is the use of HAT medium (hypoxanthine, aminopterin, and thymidine) (Sigma Chemical Co., St. Louis, Mo.). After about seven days, the resulting fused cells or hybridomas may be screened in order to determine the presence of antibodies which recognize the desired antigen. Following several clonal dilutions and reassays, hybridoma producing antibodies that bind to the protein of interest can be isolated.

[0132] Other techniques may also be utilized to construct monoclonal antibodies. (See Huse et al., "Generation of a Large Combinational Library of the Immunoglobulin Repertoire in Phage Lambda," Science 246:1275-1281, 1989; see also Sastry et al., "Cloning of the Immunological Repertoire in Escherichia coli for Generation of Monoclonal Catalytic Antibodies: Construction of a Heavy Chain Variable Region-Specific cDNA Library," Proc. Natl. Acad Sci. USA 86:5728-5732, 1989; see also Alting-Mees et al., "Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas," Strategies in Molecular Biology 3:1-9, 1990; these references describe a commercial system available from Stratagene, La Jolla, Calif., which enables the production of antibodies through recombinant techniques.) Briefly, mRNA is isolated from a B cell population and utilized to create heavy and light chain immunoglobulin cDNA expression libraries in the .lamda.IMMUNOZAP(H) and .lamda.IMMUNOZAP(L) vectors. These vectors may be screened individually or co-expressed to form Fab fragments or antibodies (see Huse et al. (supra); see also Sastry et al. (supra)).

[0133] Similarly, binding partners can also be constructed utilizing recombinant DNA techniques to incorporate the variable regions of a gene that encodes a specifically binding antibody. The construction of these binding partners can be readily accomplished by one of ordinary skill in the art given the disclosure provided herein. (See Larrick et al., "Polymerase Chain Reaction Using Mixed Primers: Cloning of Human Monoclonal Antibody Variable Region Genes From Single Hybridoma Cells," Biotechnology 7:934-938, 1989; Riechmann et al., "Reshaping Human Antibodies for Therapy," Nature 332:323-327, 1988; Roberts et al., "Generation of an Antibody with Enhanced Affinity and Specificity for its Antigen by Protein Engineering," Nature 328:731-734, 1987; Verhoeyen et al., "Reshaping Human Antibodies: Grafting an Antilysozyme Activity," Science 239:1534-1536, 1988; Chaudhary et al., "A Recombinant Immunotoxin Consisting of Two Antibody Variable Domains Fused to Pseudomonas Exotoxin," Nature 339:394-397, 1989; see also U.S. Pat. No. 5,132,405 entitled "Biosynthetic Antibody Binding Sites." ) Briefly, in one embodiment, DNA segments encoding the desired protein or peptide of interest-specific antigen binding domains are amplified from hybridomas that produce a specifically binding monoclonal antibody, and are inserted directly into the genome of a cell that produces human antibodies. (See Verhoeyen et al. (supra); see also Reichmann et al. (supra)). This technique allows the antigen-binding site of a specifically binding mouse or rat monoclonal antibody to be transferred into a human antibody. Such antibodies are preferable for therapeutic use in humans because they are not as antigenic as rat or mouse antibodies.

[0134] In an alternative embodiment, genes that encode the variable region from a hybridoma producing a monoclonal antibody of interest are amplified using oligonucleotide primers for the variable region. These primers may be synthesized by one of ordinary skill in the art, or may be purchased from commercially available sources. For instance, primers for mouse and human variable regions including, among others, primers for V.sub.Ha, V.sub.Hb, V.sub.Hc, V.sub.Hd, C.sub.Hl, V.sub.L and C.sub.L regions, are available from Stratagene (La Jolla, Calif.). These primers may be utilized to amplify heavy or light chain variable regions, which may then be inserted into vectors such as IMMUNOZAP.TM.(H) or IMMUNOZAP.TM.(L) (Stratagene), respectively. These vectors may then be introduced into E. coli for expression. Utilizing these techniques, large amounts of a single-chain polypeptide containing a fusion of the V.sub.H and V.sub.L domains may be produced (see Bird et al., Science 242:423-426, 1988).

[0135] Monoclonal antibodies and other binding partners can be produced in a number of host systems, including tissue cultures, bacteria, eukaryotic cells, plants and other host systems known in the art.

[0136] Once suitable antibodies or binding partners have been obtained, they may be isolated or purified by many techniques well known to those of ordinary skill in the art (see Antibodies: A Laboratory Manual, Harlow and Lane (supra)). Suitable techniques include peptide or protein affinity columns, HPLC or RP-HPLC, purification on protein A or protein G columns, or any combination of these techniques. Within the context of the present invention, the term "isolated" as used to define antibodies or binding partners means "substantially free of other blood components."

[0137] The binding partners of the present invention have many uses. For example, antibodies can be utilized in flow cytometry to identify cells bearing such a protein. Briefly, in order to detect the protein or peptide of interest on cells, the cells are incubated with a labeled monoclonal antibody which specifically binds to the protein of interest, followed by detection of the presence of bound antibody. Labels suitable for use within the present invention are well known in the art including, among others, flourescein isothiocyanate (FITC), phycoerythrin (PE), horse radish peroxidase (HRP), and colloidal gold. Particularly preferred for use in flow cytometry is FITC, which may be conjugated to purified antibody according to the method of Keltkamp in "Conjugation of Fluorescein Isothiocyanate to Antibodies. I. Experiments on the Conditions of Conjugation," Immunology 18:865-873, 1970. (See also Keltkamp, "Conjugation of Fluorescein Isothiocyanate to Antibodies. II. A Reproducible Method," Immunology 18:875-881, 1970; Goding, "Conjugation of Antibodies with Fluorochromes: Modification to the Standard Methods," J. Immunol. Methods 13:215-226, 1970.) The antibodies can also be used to target drugs to Neisseria meningitidis, Aspergillus fumigatus or Candida glabrata as well as a diagnostic for determining infection by these organisms.

D. Assays That Utilize the Hsp Polypeptides, or Antibodies Thereto, of the Present Invention

[0138] A variety of assays can be utilized in order to detect the Hsp polypeptides from Neisseria meningitidis, Aspergillus fumigatus or Candida glabrata of the present invention, or antibodies that specifically bind to such Hsp polypeptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: countercurrent immuno-electrophoresis (CIEP), radioimmunoassays, radioimmunoprecipitations, enzyme-linked immuno-sorbent assays (ELISA), dot blot assays, inhibition or competition assays, and sandwich assays, immunostick (dipstick) assays, simultaneous immunoassays, immunochromatographic assays, immunofiltration assays, latex bead agglutination assays, immunofluorescent assays, biosensor assays, and low-light detection assays (see U.S. Pat. Nos. 4,376,110 and 4,486,530; see also Antibodies: A Laboratory Manual (supra).

[0139] A fluorescent antibody test (FA-test) uses a fluorescently labeled antibody able to bind to one of the proteins of the invention. For detection, visual determinations are made by a technician using fluorescence microscopy, yielding a qualitative result. In one embodiment, this assay is used for the examination of tissue samples or histological sections.

[0140] In latex bead agglutination assays, antibodies to one or more of the proteins of the present invention are conjugated to latex beads. The antibodies conjugated to the latex beads are then contacted with a sample under conditions permitting the antibodies to bind to desired proteins in the sample, if any. The results are then read visually, yielding a qualitative result. In one embodiment, this format can be used in the field for on-site testing.

[0141] Enzyme immunoassays (EIA) include a number of different assays able to utilize the antibodies provided by the present invention. For example, a heterogeneous indirect EIA uses a solid phase coupled with an antibody of the invention and an affinity purified, anti-IgG immunoglobulin preparation. Preferably, the solid phase is a polystyrene microtiter plate. The antibodies and immunoglobulin preparation are then contacted with the sample under conditions permitting antibody binding, which conditions are well known in the art. The results of such an assay can be read visually, but are preferably read using a spectrophotometer, such as an ELISA plate reader, to yield a quantitative result. An alternative solid phase EIA format includes plastic-coated ferrous metal beads able to be moved during the procedures of the assay by means of a magnet. Yet another alternative is a low-light detection immunoassay format. In this highly sensitive format, the light emission produced by appropriately labeled bound antibodies are quantitated automatically. Preferably, the reaction is performed using microtiter plates.

[0142] In an alternative embodiment, a radioactive tracer is substituted for the enzyme mediated detection in an EIA to produce a radioimmunoassay (RIA).

[0143] In a capture-antibody sandwich enzyme assay, the desired protein is bound between an antibody attached to a solid phase, preferably a polystyrene microtiter plate, and a labeled antibody. Preferably, the results are measured using a spectrophotometer, such as an ELISA plate reader.

[0144] In a sequential assay format, reagents are allowed to incubate with the capture antibody in a step wise fashion. The test sample is first incubated with the capture antibody. Following a wash step, an incubation with the labeled antibody occurs. In a simultaneous assay, the two incubation periods described in the sequential assay are combined. This eliminates one incubation period plus a wash step.

[0145] A dipstick/immunostick format is essentially an immunoassay except that the solid phase, instead of being a polystyrene microtiter plate, is a polystyrene paddle or dipstick. Reagents are the same and the format can either be simultaneous or sequential.

[0146] In a chromatographic strip test format, a capture antibody and a labeled antibody are dried onto a chromatographic strip, which is typically nitrocellulose or nylon of high porosity bonded to cellulose acetate. The capture antibody is usually spray dried as a line at one end of the strip. At this end there is an absorbent material that is in contact with the strip. At the other end of the strip the labeled antibody is deposited in a manner that prevents it from being absorbed into the membrane. Usually, the label attached to the antibody is a latex bead or colloidal gold. The assay may be initiated by applying the sample immediately in front of the labeled antibody.

[0147] Immunofiltration/immunoconcentration formats combine a large solid phase surface with directional flow of sample/reagents, which concentrates and accelerates the binding of antigen to antibody. In a preferred format, the test sample is preincubated with a labeled antibody then applied to a solid phase such as fiber filters or nitrocellulose membranes or the like. The solid phase can also be precoated with latex or glass beads coated with capture antibody. Detection of analyte is the same as standard immunoassay. The flow of sample/reagents can be modulated by either vacuum or the wicking action of an underlying absorbent material.

[0148] A threshold biosensor assay is a sensitive, instrumented assay amenable to screening large numbers of samples at low cost. In one embodiment, such an assay comprises the use of light addressable potentiometric sensors wherein the reaction involves the detection of a pH change due to binding of the desired protein by capture antibodies, bridging antibodies and urease-conjugated antibodies. Upon binding, a pH change is effected that is measurable by translation into electrical potential (.mu.volts). The assay typically occurs in a very small reaction volume, and is very sensitive. Moreover, the reported detection limit of the assay is 1,000 molecules of urease per minute.

[0149] The present invention also provides for probes and primers for detecting Neisseria meningitidis, Aspergillus fumigatus and Candida glabrata.

[0150] In one embodiment of this aspect of the invention, probes are provided that are capable of specifically hybridizing to Neisseria meningitidis, Aspergillus fumigatus or Candida glabrata Hsp genes DNA or RNA. For purposes of the present invention, probes are "capable of hybridizing" to Neisseria meningitidis, Aspergillus fumigatus or Candida glabrata Hsp gene DNA or RNA if they hybridize under conditions of high stringency (see Sambrook et al. (supra)). Preferably, the probe may be utilized to hybridize to suitable nucleotide sequences under highly stringent conditions, such as 6.times.SSC, 1.times. Denhardt's solution (Sambrook et al. (supra)), 0.1% SDS at 65.degree. C. and at least one wash to remove excess probe in the presence of 0.2.times.SSC, lx Denhardt's solution, 0.1% SDS at 65.degree. C. Except as otherwise provided herein, probe sequences are designed to allow hybridization to Neisseria meningitidis, Aspergillus fumigatus or Candida glabrata DNA or RNA sequences, but not to DNA or RNA sequences from other organisms, particularly other bacterial and fungal sequences. The probes are used, for example, to hybridize to nucleic acids that have been isolated from a test sample. The hybridized probe is then detected, thereby indicating the presence of the desired cellular nucleic acid. Preferably, the cellular nucleic acid is subjected to an amplification procedure, such as PCR, prior to hybridization.

[0151] Probes of the present invention may be composed of either deoxyribonucleic acids (DNA) or ribonucleic acids (RNA), and may be as few as about 12 nucleotides in length or more typically about 18 to 24 nucleotides or longer comprising a sequence derived from a fragment of the sequences of the Neisseria meningitidis, Aspergillus fumigatus or Candida glabrata Hsp genes provided by this invention. As used herein, a sequence is "derived from a fragment" when it contains a nucleotide sequences identical to a contiguous nucleotide sequence present in the fragment, or contains a nucleotide sequence that results from reading errors that occur during a PCR amplification of the fragment, or contains a degenerate nucleotide sequence that encodes an amino acid sequence that is identical to or has conservative substations of an amino acid sequence encoded by the fragment. Selection of probe size is somewhat dependent upon the use of the probe, and is within the skill of the art.

[0152] Suitable probes can be constructed and labeled using techniques that are well known in the art. Shorter probes of, for example, 12 bases can be generated synthetically. Longer probes of about 75 bases to less than 1.5 kb are preferably generated by, for example, PCR amplification in the presence of labeled precursors such as [.alpha.-.sup.32P]dCTP, digoxigenin-dUTP, or biotin-dATP. Probes of more than 1.5 kb are generally most easily amplified by transfecting a cell with a plasmid containing the relevant probe, growing the transfected cell into large quantities, and purifying the relevant sequence from the transfected cells. (See Sambrook et al. (supra)).

[0153] Probes can be labeled by a variety of markers, including for example, radioactive markers, fluorescent markers, enzymatic markers, and chromogenic markers. The use of .sup.32P is particularly preferred for marking or labeling a particular probe.

[0154] It is a feature of this aspect of the invention that the probes can be utilized to detect the presence of Neisseria meningitidis, Aspergillus fumigatus or Candida glabrata Hsp mRNA or DNA within a sample. However, if the organisms are present in only a limited number, then it may be beneficial to amplify the relevant sequence such that it may be more readily detected or obtained.

[0155] A variety of methods may be utilized in order to amplify a selected sequence, including, for example, RNA amplification (see Lizardi et al., Bio/Technology 6:1197-1202, 1988; Kramer et al., Nature 339:401-402, 1989; Lomeli et al., Clinical Chem. 35(9):1826-1831, 1989; U.S. Pat. No. 4,786,600), and DNA amplification utilizing LCR or Polymerase Chain Reaction ("PCR") (see U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; see also U.S. Pat. Nos. 4,876,187 and 5,011,769, which describe an alternative detection/amplification system comprising the use of scissile linkages), or other nucleic acid amplification procedures that are well within the level of ordinary skill in the art. With respect to PCR, for example, the method may be modified as known in the art. PCR may also be used in combination with reverse dot blot hybridization (Iida et al., FEMS Microbiol Lett. 114.167-172, 1993). PCR products may be quantitatively analyzed by incorporation of dUTP (Duplaa et al., Anal. Biochem. 212:229-236, 1993), and samples may be filter sampled for PCR-gene probe detection (Bej et al., Appl. Environ. Microbiol. 57:3529-3534, 1991).

[0156] Within a preferred embodiment, PCR amplification is utilized to detect Neisseria meningitidis, Aspergillus fumigatus or Candida glabrata Hsp DNA. Briefly a DNA sample is denatured at 95.degree. C. in order to generate single-stranded DNA. Specific primers are then annealed to the single-stranded DNA at 37.degree. C. to 70.degree. C., depending on the proportion of AT/GC in the primers. The primers are extended at 72.degree. C. with Taq DNA polymerase in order to generate the opposite strand to the template. These steps constitute one cycle, which may be repeated in order to amplify the selected sequence.

[0157] Within an alternative preferred embodiment, LCR amplification is utilized for amplification. LCR primers are synthesized such that the 5' base of the upstream primer is capable of hybridizing to a unique sequence in a desired gene to specifically detect a strain of Neisseria meningitidis, Aspergillus fumigatus or Candida glabrata harboring the desired gene.

[0158] Within another preferred embodiment, the probes are used in an automated, non-isotopic strategy wherein target nucleic acid sequences are amplified by PCR, and then desired products are determined by a colorimetric oligonucleotide ligation assay (OLA) (Nickerson et al., Proc. Natl. Acad. Sci. USA 81:8923-8927, 1990).

[0159] Primers for the amplification of a selected sequence should be selected from sequences that are highly specific and form stable duplexes with the target sequence. The primers should also be non-complementary, especially at the 3' end, should not form dimers with themselves or other primers, and should not form secondary structures or duplexes with other regions of DNA. In general, primers of about 18 to 20 nucleotides are preferred, and can be easily synthesized using techniques well known in the art. PCR products, and other nucleic acid amplification products, may be quantitated using techniques known in the art (Duplaa et al., Anal. Biochem. 212:229-236, 1993; Higuchi et al., Biol. Technology 11: 1026-1030).

[0160] Further a biochip array specific for Neisseria meningitidis, Aspergillus fumigatus or Candida glabrata, comprised of a substrate to which either oligonucleotides, polypeptides or antibodies may be bound can be manufactured using the invention disclosed herein in combination with current biochip technologies. U.S. Pat. No. 5,445,934. By using such a substrate with oligonucleotides derived from the Hsp sequences of this invention or antibodies specific for the Hsp gene products of this invention, a high throughput screening tool can be created to identify the specific Neisseria, Aspergillus or Candida species in many samples.

E. Pharmaceutical Compositions and Methods

[0161] By administering a Neisserial, Aspergillal or Candidal Hsp to an animal, the respective Hsp can induce an immune response in the animal to Neisseria, Aspergillus or Candida species, respectively, preferably providing resistance to such bacterial or fungal infection. Accordingly, the isolation of Neisserial, Aspergillal and Candidal Hsp genes and polypeptides of the present invention provides a platform for the generation of compositions containing isolated polypeptides, fragments or variants of Hsps that are useful in diagnosis and treatment of Neisserial, Aspergillal or Candidal associated disorders. As used herein, "treatment" means to administer an agent that prevents or reduces the severity of a disorder caused by an infection by a Neisseria, Aspergillus or Candida species.

[0162] Therefore, another aspect of the present invention provides compositions and methods comprising one or more of the above-described Hsp polypeptides or antibodies to Hsps in combination with one or more pharmaceutically or physiologically acceptable carriers, adjuvants, binders or diluents. Such compositions can be used to elicit or enhance an immune response, while antibodies can be used to block progression of disease in a recipient animal, which is preferably a human being, and preferably elicits or enhances a protective or partially protective immunity against Neisseria meningitidis, Aspergillus fumigatus or Candida glabrata, or against an organism that is targeted by an antigen fused to an Hsp of the present invention.

[0163] Preferably, such carriers, adjuvants, binders or diluents are nontoxic to recipients at the dosages and concentrations employed. Ordinarily, the preparation of such compositions entails combining the isolated Hsp polypeptide of this invention with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with nonspecific serum albumin are exemplary appropriate diluents. Examples of adjuvants include alum or aluminum hydroxide for humans.

[0164] It will be evident in light of the present specification to those in the art that the amount and frequency of administration can be optimized in clinical trials, and will depend upon such factors as the disease or disorder to be treated, the degree of immune inducement, enhancement, or protection required, and many other factors.

[0165] In one embodiment, the composition is administered orally, and the purified Hsp of the invention is taken up by cells, such as cells located in the lumen of the gut. Alternatively, the Hsp composition can be parenterally administrated via the subcutaneous route, or via other parenteral routes. Other routes include buccal/sublingual, rectal, nasal, topical (such as transdermal and ophthalmic), vaginal, pulmonary, intraarterial, intramuscular, intraperitoneal, intraocular, intranasal or intravenous, or indirectly. The Hsp compositions of the present invention can be prepared and administered as a liquid solution, or prepared as a solid form (e.g., lyophilized) which can be administered in solid form or resuspended in a solution in conjunction with administration.

[0166] Depending upon the application, quantities of injected Hsp in the composition will vary generally from about 0.1 .mu.g to 1000 mg, typically from about 1 .mu.g to 100 mg, preferably from about 10 .mu.g to 10 mg, and preferably from about 100 .mu.g to 1 mg, in combination with the physiologically acceptable carrier, binder or diluent. Booster immunizations can be given from 2-6 weeks later.

[0167] The pharmaceutical compositions of the present invention may be placed within containers, along with packaging material, preferably consumer-acceptable, which provides instructions regarding the use of such pharmaceutical compositions, to provide kits suitable for use within the present invention. Generally, such instructions will include a tangible expression describing the reagent concentration, as well as within certain embodiments, relative amounts of excipient ingredients or diluents (e.g., water, saline or PBS) which may be necessary to reconstitute the pharmaceutical composition.

[0168] The Hsp gene products of this invention may also be used as immunological carriers in conjugate vaccines. Hsps are beneficial carriers of antigens because, unlike other carriers, they do not have an immunosuppressive effect. See Barrios et al., Eur. J Immunol. 22:1365-1372, 1992; Suzue and Young, in Stress-Inducible Cellular Responses 77:451-465, 1996 (edited by U. Feige et al.). Such carriers may be used to elicit an increased immune response to the conjugated molecule. Alternatively, small Hsp peptides or polypeptides containing antigenic epitopes derived from the larger Hsp polypeptides provided herein, can be conjugated or fused to other carrier proteins to elicit an immunogenic response to the small Hsp antigenic epitope. The Hsp gene products of this invention may therefore be used (in conjugates or fusion proteins) as carriers to elicit an immunogenic response against other target antigens, or as antigens to elicit an immunogenic response against epitopes present on the Hsps.

[0169] As used herein, a "fusion protein" is a protein comprised of a Hsp polypeptide, or portion thereof, which is has a peptide bond linkage with an amino acid sequence of an additional polypeptide chain such that a single polypeptide chain is formed which contains an amino acid sequence derived from the Hsp joined with an amino acid sequence derived from the additional polypeptide chain. In one example of typical fusion proteins, a Hsp polypeptide or portion thereof is fused to an additional carrier polypeptide that enhances an immunogenic response in an animal. Example of such polypeptides include but are not limited to keyhole limpet hemocyanin, (KLH) bovine gamma globulin (BGG), serum albumin from various animals (SA) and polypeptides that provide antigenic determinants in addition to that provided by a single Hsp domain. Additional antigenic determinants may include for example, polypeptide domains from more than one Hsp and/or multiple duplications of an antigenic polypeptide domain from a single Hsp. In these cases the target of the immune response of interest is typically the Hsp portion of the fusion protein. Alternatively, a Hsp polypeptide of the present invention can serve as the carrier portion of a fusion polypeptide when the additional polypeptide to which it is fused is the target antigen for eliciting an immunogenic response.

[0170] One method for producing a fusion protein is by in frame ligation of a nucleic acid sequence encoding a Hsp with a nucleic acid sequence encoding an additional polypeptide to form a hybrid sequence. The hybrid sequence is inserted into an expression vector to form a construct having the hybrid fragment under the control of a promoter operably linked thereto. The construct is introduced into a suitable host cell capable of expressing the hybrid fragment from the vector sequence. Upon expression, the fusion protein is produced and may be isolated from the host cell. When the host cell is a bacterium, the fusion protein may aggregate into inclusion bodies and be readily isolated using methods well known in the art. Alternatively, the fusion protein may include signaling sequences selected to direct the fusion protein to export from the cell into the extracellular medium in which the host cell is cultured.

[0171] A further aspect of the present invention is protection from Neisserial, Aspergillal or Candidal associated diseases by either immunization with the Hsp gene products of the present invention (e.g. by intramuscular injection of an expression vector containing an Hsp gene) or by using gene transfer techniques to deliver a vector containing Hsp genes or fragments thereof to be expressed within the cells of the animal. The compositions and methodologies described herein are suitable for a variety of uses. To this end, the following examples are presented for purposes of illustration, not limitation.

EXAMPLES

Example 1

Cloning of an Internal Fragment of the Neisseria Meningitidis HSP70 Gene

[0172] Comparison of previously characterized bacterial Hsp70 (or DnaK) proteins was used to identify conserved regions and design degenerate primers suitable for PCR-amplification of an internal region of the unknown Neisseria meningitidis Hsp70 gene.

[0173] Forward degenerate primer W247 corresponded to a sequence encoding amino acids 145-150 of the consensus Hsp70 sequence (PAYFND; SEQ ID NO:29). TABLE-US-00001 W247: 5'-CCNGCNTAYTTYAAYGAY-3' (SEQ ID NO: 30)

[0174] Reverse degenerate primer W248 was complementary to a sequence encoding amino acids 476-482 of the consensus Hsp70 sequence (PQIEVTF; SEQ ID NO:31). TABLE-US-00002 W248: 5'-RAANGTNACYTCDATYTGNGG-3' (SEQ ID NO: 32)

[0175] Note that in all sequences provided herein A corresponds to adenosine, C to cytidine, G to guanosine, T to thymidine, I to inosine, R to A or G, Y to C or T, N to A, C, T or G, K to G or T, M to A or C, S to G or C, W to T or A, B to C, G or T, D to A, G or T, H to A, C or T, and V to A, C or G. Unless specified, all molecular DNA manipulations (plasmid isolation, restriction enzyme digestion, ligation, etc.) were carried on under standard conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1989.

[0176] PCR reactions were carried out according to Perkin-Elmer's recommendations. All reagents used for PCR reactions were supplied by Perkin-Elmer unless indicated otherwise. Reaction mixtures (total volume of 100 ul) contained 0.5 to 1 ug of genomic DNA, 100 pmoles of each of the degenerate primers (W247 and W248, synthesized by Life Technologies), 500 uM each of dNTPs (New England BioLabs), 1.times. PCR buffer, 4 mM MgSO4, and 1.25 units of Taq polymerase. Reactions were incubated at 95.degree. C. for 30 seconds, at 51.degree. C. for 3 minutes and then at 72.degree. C. for 1 minute. After repeating the above cycle for a total of 40 times, reactions were incubated at 72.degree. C. for an additional 4 minutes. Genomic DNA from Neisseria meningitidis (ATCC 13090) was obtained from Dr. Lee Weber (University of Nevada, Reno, Nev., USA).

[0177] A PCR fragment of about one kbp in length was isolated from a low-melting point agarose gel by phenol extraction and ligated into pCR2.1 TA cloning vector (Invitrogen) using standard conditions. The ligation reaction was used to transform E. coli DH5a cells, and transformants were selected on LB agar plates with kanamycin D. Recombinant plasmids were identified after digestion of plasmid DNA with EcoRI restriction enzyme.

[0178] Plasmids containing the above fragment of Neisseria meningitidis Hsp70 gene were subjected to DNA sequencing using the dye-terminator method on a Prizm 310 automatic sequencer (ABI). Sequence data were assembled and analyzed using DNA Star (DNASTAR Inc.) as well as DNA Strider (CEA, France) software. E. coli dnaK gene and protein sequences available from GenBank were used for comparison purposes during assembly.

[0179] Three clones originating from a mixture of three separate PCR reactions were sequenced using M13 forward and reverse universal primers: TABLE-US-00003 M13F: 5'-GTAAAACGACGGCCAG-3' (SEQ ID NO: 33) M13R: 5'-CAGGAAACAGCTATGAC-3' (SEQ ID NO: 34)

[0180] Sequences obtained were used to design an additional pair of primers for sequencing: TABLE-US-00004 N1: 5'-CTGCCGTACATCACCATGG-3' (SEQ ID NO: 25) N2: 5'-GGCTTCTTGTACTTTCGGC-3' (SEQ ID NO: 26)

[0181] FIG. 1 shows the strategy for sequencing the internal Neisseria meningitidis Hsp70 gene fragment.

[0182] FIG. 2 lists the DNA sequence of this region (assembled from information obtained from sequencing three Hsp70 gene fragment-containing plasmids).

Example 2

Cloning the Ends of the Neisseria Meningitidis Hsp70 Gene by Inverse PCR

[0183] The so called inverse PCR approach was used to clone missing ends of the Neisseria meningitidis Hsp70 gene. From the restriction map of the assembled partial DNA sequence BamHI, EcoRI, HincII and Hind III were chosen as enzymes that do not cut.

[0184] Approximately 2 ug of Neisseria meningitidis genomic DNA were digested with each of the above enzymes, phenol-extracted, precipitated with ethanol and dissolved in 1.times. ligation buffer at approx. 80 ng/ml. Fragments were ligated and then used as templates for PCR amplification (40 cycles of 1 minute at 95.degree. C., 2 minutes at 65.degree. C. and 2 minutes at 72.degree. C.) in reaction mixtures (as described above) containing primers nested near the ends of known sequence and pointing outside: TABLE-US-00005 N70-5: 5'-GGTCGGCTCGTTGATGATGCGTTTCAC-3' (SEQ ID NO: 35) N70-3: 5'-GCTTCTGCCAACAAATGTTTGGGTCAG-3' (SEQ ID NO: 36)

[0185] Only DNA digested with HindIII seemed to produce a specific PCR-generated band after amplification. The 0.9 kbp-long fragment was purified from a low-melting point agarose gel and cloned into pCR2.1 vector. A recombinant containing the fragment was identified, and the inserted fragment was sequenced using M13F and M13R primers. It turned out that cloned fragment had been amplified making use of only the N70-5 primer. Still, it represented a region from the 5' end of the Neisseria meningitidis Hsp70 gene. Unfortunately, only 44 nucleotides of new sequence could be determined because a HindIII site happened to be present just upstream from the 5'-end of the previously sequenced internal Hsp70 gene fragment. Nevertheless, the sequence of the fragment allowed the resolution of ambiguities created by the use of the degenerate W247 primer. See region B in FIG. 3 as well as the sequence in FIG. 4.

[0186] A Neisseria meningitidis (ATCC 13090) genomic library in bacteriophage lambda was prepared using routine procedures and was screened using the above-described internal region of the Neisseria meningitidis Hsp70 gene as the probe. A recombinant clone containing the Neisseria meningitidis Hsp70 gene was used as template for inverse PCR. Additional primers (pointing towards the interior of the known Hsp70 sequence) were designed near the known ends of the internal Hsp70 fragment in regions not interrupted by RsaI restriction sites. Recombinant phage DNA was digested with RsaI, and resulting fragments were circularized as above.

[0187] To amplify the 5'-end region, primers N70-5 (see above) and N70-5B were used for PCR amplification of the ligation reaction: TABLE-US-00006 N70-5B: 5'-GCCGGTTTGGCATTCGTTATGGAC-3' (SEQ ID NO: 37)

[0188] To amplify the 3'-end region, primers N70-3 (see above) and N70-3B were used for PCR of the ligation reaction: TABLE-US-00007 N70-3B: 5'-GCGTTCGCGTTCGCCTTGCAGTAC-3' (SEQ ID NO: 38)

[0189] PCR reactions were carried out as described before. PCR products were isolated from low-melting point agarose gels and inserted into vector pCR2.1 as also described before.

[0190] Sequencing of cloned PCR products revealed the complete sequence of the 3' end of the Neisseria meningitidis Hsp70 gene (and clarification of ambiguities resulting from the use of degenerate primer W247 in the cloning of the internal Hsp70 region) as well as 3'-untranslated sequence (region E in FIG. 3). The nucleotide sequence of the 5' end of the Hsp70 gene was also established (region C in FIG. 3), except for the first 28 bp (which had been removed by RsaI digestion).

[0191] To determine the nucleotide sequence at the 5' end of the Hsp70 gene, recombinant phage DNA was digested with restriction enzyme Sau3A, fragments were circularized as before, and the ligation reaction was subjected to PCR using a set of primers located between 5' end of the known Hsp70 sequence and closest internal Sau3A site: TABLE-US-00008 N70-5C: 5'-TTCCGAAAACGGTCAAAC-3' (SEQ ID NO: 39) N70-5D: 5'-ATGGCCAAACAAGAGTTG-3' (SEQ ID NO: 40)

[0192] PCR reaction, isolation of PCR product from a low-melting point agarose gel and ligation into vector pCR2.1 were carried out as described before. The ligation reaction was then PCR-amplified using M13F and N70-5C primers. The resulting PCR product was then purified from an agarose gel using a gel extraction kit from Qiagen and used directly for sequencing using the T7PROM primer. This protocol produced the complete nucleotide of the 5'-end of the Neisseria meningitidis Hsp70 gene as well as 5'-untranslated and promoter sequences (region D in FIG. 3).

[0193] FIG. 4 lists the complete nucleotide sequence of the Neisseria meningitidis Hsp70 gene as well as of flanking regions. The derived amino acid sequence of the protein product of the gene is also shown.

Example 3

Nesisseria Meningitidis Hsp70 Expression Vectors

[0194] To clone the Neisseria meningitidis Hsp70-coding region, DNA from a recombinant bacteriophage containing the Hsp70 gene served as the template in a PCR amplification reaction that included primers N70-M and N70-Z, complementary to sequence at the 5'-end (including an NdeI site) and the 3'-end of the Hsp70 gene, respectively. TABLE-US-00009 N70-M: 5'-TACATATGGCAAAAGTAATCGGTATC-3' (SEQ ID NO: 41) N70-Z: 5'-TTTATTTTTTGTCGTCTTTTAC-3' (SEQ ID NO: 42)

[0195] PCR product was purified from an agarose gel using a gel extraction kit (Qiagen) and ligated into pCR2.1 vector. Two positive clones were identified by EcoRI digestion of miniprep DNA isolated from E. coli DH5a colonies resistant to kanamycin D and further confirmed by restriction analysis using HindIII, NotI, NdeI and ClaI. Inserted DNA was then sequenced using primers M13F, M13R, N1, N2, N70-5, N70-5B, N70-5C, N70-3, N70-3B, as well as new primer N10: TABLE-US-00010 N10: 5'-GTCCAAATAAGCGATAACG-3' (SEQ ID NO: 43)

[0196] FIG. 5 illustrates the sequencing strategy employed.

[0197] The sequence obtained (FIG. 6) differed from that presented in FIG. 3 by an A instead of a G at positions 1528 (counted from the NdeI recognition site) and 1647. Only the first of these differences would also be reflected at the protein level. Because sequence comparisons showed that residue 509 is typically serine, the sequence presented in FIG. 6 was assumed to be the correct sequence.

[0198] An NdeI-EcoRI (site located downstream from the stop codon) fragment of the above pCR2.1-based plasmid including the complete Hsp70-coding sequence was inserted in between the NdeI and EcoRI sites of pET24A+and pET28A+T7 expression vectors. Positive clones were identified by digestion of DNA isolated from kanamycin resistant transformed DH5a colonies with NdeI and EcoRI and electrophoretic analysis. Single positive clones from each set was sequenced using primers T7PROM, T7TERM, N1, N2, N70-5, N70-5B, N70-5C, N70-3B and new primers TABLE-US-00011 N20:.sup.1 5'-GCCGCCAAACGTTTGATC-3', (SEQ ID NO: 44) N21: 5'-ACCATGGGCGGCGTGATG-3', (SEQ ID NO: 45) N22: 5'-GAAGCCAATGCCGAGGAA-3', (SEQ ID NO: 46) N23: 5'-TGCGTCGCCGTTGTTGGC-3', (SEQ ID NO: 47) N24: 5'-GGTATCGCCGTTGGTTGC-3', (SEQ ID NO: 48) and N25: 5'-GAGTTTGTCGCCGTAGTC-3'. (SEQ ID NO: 49)

[0199] With these additional primers, both strands of the Neisseria meningitidis Hsp70 gene could be sequenced in their entireties. The sequencing strategy employed is illustrated in FIG. 7.

[0200] FIG. 8 lists the DNA sequence of the Neisseria meningitidis Hsp70 gene in pET24A+vector (pETN70), and FIG. 9 shows the sequence of its histidine-tagged derivative in pET28A+vector (pETN70H).

[0201] The functionality of both expression plasmids was confirmed by transformation into E. coli BL2 1 (DE3) cells and detection of the expected protein band after induction with IPTG (1 mM) in small cultures (2.times. YT medium supplemented with kanamycin D). See FIG. 10.

Example 4

Purification and Characterization of Recominant Neisseria Meningitidis Hsp70 Protein

[0202] E. coli BL21(DE3) bacteria transformed with pETN70H were grown in 2.times. YT medium supplemented with 30 mg of kanamycin D at 37.degree. C. to OD600 nm approx. 0.5-0.8 and then induced with 0.5 mM IPTG for 3 hours. Cultures were then chilled on ice, bacteria collected by centrifugation at 6000 rpm at 4.degree. C. for 5 minutes and pellets were frozen at -80.degree. C.

[0203] Frozen bacterial pellet from a 4 liter culture was crushed, transferred to a blender, and blended in 200 ml of 6M GuHCl, 50 mM Tris-HCl pH7.5, 0.5mM beta-mercaptoethanol. Lysate was cleared by centrifugation at 8000 rpm at 4.degree. C. for 15 minutes, and the supernatant solution was mixed overnight at room temperature with approximately 40 ml of slurry containing 2 0ml of Ni-Sepharose (Chelating Sepharose, Pharmacia) equilibrated with 6M GuHCl, 50 mM Tris-HCl pH7.5, 0.5 mM beta-mercaptoethanol.

[0204] Resin was washed on filter paper with approximately 100 ml 6M GuHCI, 50 mM Tris-HCl pH7.5, 0.5 mM beta-mercaptoethanol, resuspended in a small volume of the same buffer and gravity-packed into a glass chromatography column (Pharmacia). The column was washed with 100 ml of buffer containing 6M GuHCl, 50 mM Tris-HCl pH7.5, 0.5 mM beta-mercaptoethanol, 1% Triton X-100. The bound protein was subjected to a buffer gradient (100 ml), beginning with the above buffer and ending with a buffer containing 1M NaCl, 50 mM Tris-HCl pH7.5, 0.5 mM beta-mercaptoethanol. The column was subsequently washed with 100 ml of 1M NaCl, 50 mM Tris-HCl pH7.5, 0.5 mM beta-mercaptoethanol and then with 100 ml of a mixture containing 5% of 1M imidazole, 0.5M NaCl, 50 mM Tris-HCl pH7.5, 0.5 mM beta-mercaptoethanol in 1M NaCl, 50 mM Tris-HCl pH7.5, 0.5 mM beta-mercaptoethanol. Finally, the column was developed with a gradient (100 ml) of 10% to 100% of IM imidazole, 0.5M NaCI, 50 mM Tris-HCl pH7.5, 0.5 mM beta-mercaptoethanol in 1M NaCl, 50 mM Tris-HCl pH7.5, 0.5 mM beta-mercaptoethanol. Fractions of 5 ml were collected. The flow rate was 4-5 ml/minute, and chromatography was monitored spectrophotometrically (A280 nm).

[0205] Fractions containing highest concentrations of recombinant protein were identified by 10% SDS-PAGE and Coomassie blue staining. An example of such an analysis is shown in FIG. 11. Appropriate fractions were pooled (usually 5-6 fractions) into a dialysis bag (12 kDa cutoff and dialyzed against three changes of 1.times.DPBS (31) at 4.degree. C. Protein solution was aliquoted and stored on ice or frozen at -80.degree. C. The concentration of the recombinant protein solution was assayed by the Lowry method.

Reactivity with Various Antibodies

[0206] Purified recombinant Neisseria meningitidis Hsp70 protein was analyzed for reactivity with following Hsp70/DnaK antibodies distributed by StressGen Biotechnologies Corp., Victoria, BC:

[0207] A) SPA-810

[0208] B) SPA-811

[0209] C) SPA-812

[0210] D) SPA-815

[0211] E) SPA-816

[0212] F) SPA-820

[0213] G) SPA-822

[0214] H) SPA-880

[0215] I) SPA-885

[0216] Samples containing 0.1 mg, 0.5 mg and/or 1 mg of recombinant protein were fractionated of 10% SDS-PAGE, electroblotted onto nitrocellulose. Blots were blocked with 5% skim milk in phosphate-buffered saline (PBS) containing 0.05% Tween20 overnight at room temperature. Then blots were incubated for I hour in the same buffer containing a 1:1000 dilution of each primary antibody. Then blots were washed 3 times (10 minutes each) with PBS, 0.05% Tween20 and incubated for I hour in 5% skim milk in PBS, 0.05% Tween20 containing a 1:1000 dilution of goat anti-rabbit IgG antibody-alkaline phosphatase (AP) conjugate (Sigma) or goat-anti-murine IgG-alkaline phosphatase (AP) conjugate (Sigma), depending on the nature of the primary antibody used. Following 3 washes in PBS, 0.05% Tween20 as above, filters were equilibrated in alkaline phosphatase reaction buffer (100 mM Tris-HCl pH9.5, 150 mM NaCl, 10 mM MgCl.sub.2) and then developed in 0.05% NBT, 0.05% BCIP in the same buffer.

[0217] Neisseria meningitidis Hsp70 was not recognized by any of the above antibodies, indicating that this protein is immunologically distinct from the homologous proteins of other microorganisms and of mammals.

Example 5

Selective Amplification of the Neisseria Meningitidis Hsp70 Gene

[0218] Development of a novel PCR-based diagnostic assay for Neisseria meningitidis would require that PCR can be used to discriminate between Hsp70 genes from Neisseria meningitidis and other microorganisms. Because bacterial meningitis is caused by Neisseria meningitidis and Streptococci, it was further of interest to demonstrate that, by using appropriate primer pairs, Hsp70 genes from both types of organisms can be detected as well as distinguished from one another.

[0219] In a first experiment, 20 ng of genomic DNA from E. coli (strain DH5alpha), Helicobacter pylori (ATCC43 504), Legionella pneumophila (ATCC3 3152), Mycobacterium tuberculosis (strain H37RV), Neisseria meningitidis (ATCC13090), Streptococcus pneumoniae (ATCC6314), or Streptococcus pyogenes (ATCC 12344) were subjected to 40 cycles of PCR under conditions described above using primers specific for the Neisseria meningitidis Hsp70 gene N1 and N2. TABLE-US-00012 N1: 5' CTGCCGTACATCACCATGG-3' (SEQ ID NO: 25) N2: 5' GGCTTCTTGTACTTTCGGG-3' (SEQ ID NO: 26)

[0220] PCR products were analyzed by electrophoresis on a 2% agarose gel in 0.5.times. TBE buffer followed by ethidium bromide staining. Only DNA from Neisseria meningitidis was specifically amplified (FIG. 12, left panel).

[0221] In a second experiment, the same DNAs were subjected to PCR using primer pair N1/N2 and Streptococcal Hsp70-specific primers W264 and W267. TABLE-US-00013 W264: 5' TGACCTTGTTGAACGTAC-3' (SEQ ID NO: 27) W267: 5' ACTTCATCAGGGTTTAC-3' (SEQ ID NO: 28)

[0222] PCR products were analyzed as before. In these reactions, a 210 bp-long fragment of Neisseria meningitidis DNA and a 179 bp-long fragment of streptococcal DNA were amplified (FIG. 12, right panel).

Example 6

Cloning of the Aspergillus Fumigatus Hsp60 Gene

[0223] Genomic DNA preparations from Aspergillus fumigatus (ATCC26933) were obtained from ATCC (Rockville, Md.).

[0224] A comparison of the few previously characterized yeast (Saccharomyces cerevisiae and Schizosaccharomyces pombe) Hsp60 genes and of representative bacterial and mammalian Hsp60 genes was used to identify conserved regions and to design degenerate primers suitable for PCR-amplification of fragments of unknown fungal Hsp60 genes.

[0225] The following primers were synthesized (Life Technologies) (orientation F-forward, R-reverse, numbers correspond to amino acids of the derived consensus Hsp60 sequence: TABLE-US-00014 F3: 5'-CCATATGAARGANYTNAARTTYGGNGT-3' (SEQ ID NO: 50) F: 37-43 F3A: 5'-AAIGAITTIAAITTTGGIGT-3' (SEQ ID NO: 51) F: 37-43 F4: 5'-CTTACATCATNCCNGGCATNCC-3' (SEQ ID NO: 52) R: 598-593 F4A: 5'-ACATCATICCIGGCATICC-3' (SEQ ID NO: 53) R: 598-593 F5: 5'-CTTAGATNCCNCCCATNCCNCCCAT-3' (SEQ ID NO: 54) R: 597-591 F5A: 5'-CATICCICCGATICCICC-3' (SEQ ID NO: 55) R: 597-592 F60-C: 5'-GCIGGIGAYGGIACIACIAC-3' (SEQ ID NO: 56) F: 118-124 F60-D: 5'-GGWCCMAAGGGHMGWAATGTYTT-3' (SEQ ID NO: 57) F: 65-72 F60-E: 5'-CCNAARATYACTAAGGAYGGTGT-3' (SEQ ID NO: 58) F: 80-87 F60-F: 5'-AARGANTTNAAATTYGGYGT-3' (SEQ ID NO: 59) F: 37-43 F60-G: 5'-TCCATNGGRTTRCANCCNGC-3' (SEQ ID NO: 60) R: 148-142 F60-H: 5'-ATNACNCCYTCYTTNCCNAC-3' (SEQ ID NO: 61) R: 211-205 F60-I: 5'-CATNCCYTCNGTNACYTC-3' (SEQ ID NO: 62) R: 230-224 F60-N1: 5'-ACYGARTGTGCYATTGTYGATGC-3' (SEQ ID NO: 63) F: 561-569 F60-N2: 5'-ACYGARGTTGCYATTGTYGATGC-3' (SEQ ID NO: 64) F: 561-569 F60-O1: 5'-TTAGTTGATGCTTCTGGTGTYGC-3' (SEQ ID NO: 65) F: 548-555 F60-O2: 5'-TTAGTTGATGCTAGYGGTGTYGC-3' (SEQ ID NO: 66) F: 548-555 F60-P: 5'-GARAARGARAARYTNCARGA-3' (SEQ ID NO: 67) F: 402-408 F60-R: 5'-GCNGCNGTNGARGARGGNAT-3' (SEQ ID NO: 68) F: 446-452 CA60-Z: 5'-TTACATGCCGCCCATGCCGCCCATACC-3' (SEQ ID NO: 69) R: 597-590 CG60-Z: 5'-TTACATCATACCTGGCATACCTGG-3' (SEQ ID NO: 70) R: 598-592

[0226] PCR reactions were carried out according to manufacturers' recommendations using AmpliTaq polymerase (Perkin-Elmer), TaqBeads (Pharmacia) or Taq polymerase (Qiagen) and a Robocycler (Stratagene). Reactions (100 ul) contained 0.5 to lug of genomic DNA of Aspergillus fumigatus (ATCC26933), 100 pmoles of each degenerate primer, 500 uM each of dNTP (New England BioLabs), 1.times.manufacturer's PCR buffer, 4 mM MgSO.sub.4, and 1.25 units of polymerase. Typically, reactions were incubated at 95.degree. C. for 30 seconds, then at 51.degree. C. for 1 minute and at 72.degree. C. for 1 minute. After repeating the above cycle for a total of 40 times, reactions were incubated at 72.degree. C. for an additional 4 minutes.

[0227] PCR-amplified fragments could be obtained with primer pairs F3/F60-I, F60-P/CA60-Z and F3/CG60-Z. The identity of these fragments was determined by sequencing their ends directly (with corresponding degenerate primers) and/or after cloning into vector pCR2.1 (Invitrogen), in which case M13 universal sequencing primers were used: TABLE-US-00015 M13F: 5'-GTAAAACGACGGCCAG-3' (SEQ ID NO: 33) M13R: 5'-CAGGAAACAGCTATGAC-3' (SEQ ID NO: 34)

[0228] Prizm310 automatic sequencer and dye-terminator technology (ABI) were used for sequencing.

[0229] The amplified fragments contained sequences from near the 5' end, from near the 3' end or from a 1.6 kbp-long central region (FIG. 13). The strategy used to sequence three independent clones containing the 1.6 kbp-long fragment is shown in FIG. 13. In addition to the Ml 3 primers, a set of custom primers was also used for sequencing: TABLE-US-00016 AF1: 5'-CCGGTGGTGATGTCACGC-3' (SEQ ID NO: 71) AF2: 5'-TTGATGACGGCAACACCG-3' (SEQ ID NO: 72) AF3: 5'-AACTCGTCGGTCAGCTTG-3' (SEQ ID NO: 73) AF4: 5'-AGAACCTCGGTGCTCGCC-3' (SEQ ID NO: 74) AF5: 5'-CGCCATGGAGCGTGTTGG-3' (SEQ ID NO: 75) AF6: 5'-TGCTGTTGAGGAGGGTAT-3' (SEQ ID NO: 76) AF7: 5'-ATGATGTCCTGAACGGCA-3' (SEQ ID NO: 77) AF8: 5'-CTGGGCGATCTTGCCGTC-3' (SEQ ID NO: 78)

[0230] Inverse PCR was then used to isolate DNA fragments containing native ends of the Aspergillus fumigatus Hsp60 gene. Based on the sequence obtained from the above fragments, the inverse PCR primers shown below were synthesized ("out" and "in" indicates primer orientation toward outside or inside of the known partial sequence, respectively). For inverse PCR of the 5'-end region, the following primers were used: TABLE-US-00017 AF60-5'in: 5'-GGTCGTAAGGTCCTTATCGAG-3' (SEQ ID NO: 79)

[0231] TABLE-US-00018 AF60-5'out: 5'-AGAGTCGAAGTCACGGCCTT-3' (SEQ ID NO: 80)

[0232] For inverse PCR of the 3'-end region, the following primers were used: TABLE-US-00019 AF60-3'in: 5'-CCTCAACAATAGCGACCTCAGT-3' (SEQ ID NO: 81) AF60-3'out: 5'-CCCCGCTGCTGCTGGCAT-3' (SEQ ID NO: 82)

[0233] Aspergillus fumigatus genomic DNA was digested with RsaI (for 5'-end inverse PCR) or Sau3A (for 3'-end inverse PCR) restriction enzymes, ligated using T4 DNA ligase and amplified with the appropriate set of inverse PCR primers. The resulting PCR fragments were isolated from gel slices using Qiagen spin columns and were sequenced directly as above using inverse primers as well as an additional sequencing primer (FIG. 13): TABLE-US-00020 AF60-5'seq: 5'-TCGGGCAGTAGTGTTCATC-3' (SEQ ID NO: 83)

[0234] The complete sequence of the Aspergillus fumigatus Hsp60 gene is shown in FIG. 14.

[0235] Comparison of the Aspergillus fumigatus Hsp60 sequence with fungal Hsp60 DNA and protein sequences available from GenBank revealed that the Aspergillus fumigatus Hsp60 gene contains two introns located at the same relative positions as introns in Histoplasma capsulatum, Paracoccidioides brasiliensis and Coccidioides immitis Hsp60 genes.

Example 7

Aspergillus Fumigatus Hsp60 Expression Plasmid

[0236] The above-discussed 1.6 kbp-long internal fragment of the Aspergillus fumigatus Hsp60 gene encompasses exon 2, intron 2 and exon 3 (except for the last few residues after the GGM repeats), and therefore has the sequence information for a protein closely resembling processed Hsp60 proteins. See Singh et al. (1990) Biochem. Biophys. Res. Commun. 169(2),391-396).

[0237] To prepare a T7 expression plasmid for production of Aspergillus fumigatus Hsp60 in bacteria, intron 2 sequences had to be removed. Exons 2 and 3 were amplified separately using primers placing the recognition sequence for restriction enzyme Earl (underlined in primer sequences) at the former intron-exon junction. PCR amplification was carried out using a pCR2.1 clone containing the 1.6 kbp Aspergillus fumigatus Hsp60 gene fragment.

[0238] Exon 2 was amplified using primers M13R and AF60-EX1R: TABLE-US-00021 (SEQ ID NO: 84) AF60-EX1R: 5' TTTCTCTTCTATCCTTGGTGATCTTAGGGGAGC-3'

[0239] Exon 3 was amplified with primers M13F and AF60-EX2F: TABLE-US-00022 (SEQ ID NO: 85) AF60-EX2F: 5'-TTTCTCTTCAGATGGTGTCTCTGTTGCCAAG-3'.

[0240] The resulting PCR fragments were purified from gel slices using Qiagen spin colunms, and the DNAs were digested with Earl. Fragments were ligated using T4 DNA ligase, and ligation products were PCR amplified using primers M13R and CG60-ZB. TABLE-US-00023 CG60-ZB: 5'-TTGGATTCTACATCATACCTGGCATAC-3' (SEQ ID NO: 86)

[0241] The resulting PCR fragment was purified as above, digested with NdeI and BamHI enzymes and ligated into NdeI-BamHI digested pET24A+ and pET28A+vectors, respectively. Recombinant plasmids, pETAF60 and pETAF60H, respectively, were identified by restriction analysis and confirmed by sequencing using the same set of AF primers that were used for sequencing the 1.6 kbp fragment (see above) and the following T7 promoter and T7 terminator primers: TABLE-US-00024 T7 promoter: 5'-TAATACGACTCACTATAGG-3' (SEQ ID NO: 87) T7 terminator: 5'-GCTAGTTATTGCTCAGCGG-3' (SEQ ID NO: 88)

[0242] FIG. 15 depicts the map of pETAF60 containing a recombinant Aspergillus fumigatus Hsp60 gene fragment. The Hsp60-coding sequence is shown in FIG. 16. FIG. 17 and FIG. 18 provide the analogous information on pETAF60H encoding a histidine-tagged Hsp60 protein.

Example 8

Expression of Recombinant Aspergillus Fumigatus Hsp60

[0243] Plasmids ETAF60 and ETAF60H were introduced into E. coil BL21(DE3) cells by transformation, and expression of fingal Hsp was examined after induction with IPTG. Data presented in FIG. 19 demonstrate induction of prominent band corresponding to a protein of approx. 57 kDa (indicated by arrowhead) in cells transformed with pETAF60. Note that the subunit molecular weight of Aspergillus fumigatus Hsp60 is predicted to be 62 kDa. The protein band induced in cells transformed with pETAF50H migrates somewhat slower, as would be expected since the encoded protein also includes a histidine tag (FIG. 19). Recombinant, histidine-tagged Aspergillus fumigatus Hsp60 was purified using a protocol similar to that described under Example 4.

Example 9

Cloning of the Candida Glabrata HSP60 Gene

[0244] Genomic DNA preparations from Candida glabrata (ATCC15545) were obtained from ATCC (Rockville, Md.).

[0245] An internal 1.6 kbp-long region of the Candida glabrata Hsp60 gene was PCR amplified from genomic DNA by AmpliTaq (Perkin-Elmer) using degenerate primers F3 and F4A. (See Example 6). Products of this reaction as well as DNA amplified in two separate reactions using primers F3 and CG60-Z were cloned into vector pCR2.1 and sequenced using primers M13F and M13R and the following custom primers: TABLE-US-00025 CG-1: 5'-CCTATGGATTTGAGAAGG-3' (SEQ ID NO: 89) CG-2: 5'-CTGATAATGTCAAGTCCC-3' (SEQ ID NO: 90) CG-3: 5'-GATCTCTTCCATCCAAGAC-3' (SEQ ID NO: 91) CG-4: 5'-GTCCTTGGAGCCGTTACC-3' (SEQ ID NO: 92) CG-5: 5'-GGTAACGGCTCCAAGGAC-3' (SEQ ID NO: 93) CG-6: 5'-GTCTTGGATGGAAGAGATC-3' (SEQ ID NO: 94) CG-8: 5'-CCTTCTCAAATCCATAGG-3' (SEQ ID NO: 95) GG-9: 5'-GGGAGTTGACATTATCAG-3' (SEQ ID NO: 96)

[0246] Inverse PCR was used to isolate DNA fragments containing the ends of the Candida glabrata Hsp60 gene. To isolate the 3'-end region, Candida glabrata genomic DNA was digested with RsaI, processed as discussed before, and amplified with CG-2 as "in" primer and the following inverse PCR "out" primer: TABLE-US-00026 CG60-3: 5'-GTTGCTTCCTTGTTGGCTACTACC-3' (SEQ ID NO: 97)

[0247] The resulting PCR fragment was isolated and cloned into pCR2.1 vector and sequenced using M13F and M13R primers.

[0248] To obtain 5'-end region of the Candida glabrata Hsp60 gene, Rsa I-digested genomic DNA was ligated and PCR amplified with following inverse PCR primers: TABLE-US-00027 CG60-5 (out): 5'-CCCCAGCGTGGCAGAGACAGCGTC-3' (SEQ ID NO: 98) CG60-5B (in): 5'-GAGAACATGGGTGCTAAGCTTCTG-3' (SEQ ID NO: 99)

[0249] The resulting PCR fragment was sequenced directly. This yielded 13 bp of additional sequence as well as the natural sequence of the Candida glabrata Hsp60 gene in a region previously derived from the degenerate primer F3 only. To obtain additional sequence from the [[5i]] 5' end, the following new primers were designed and used to amplify MspI digested and ligated genomic DNA: TABLE-US-00028 CG60-F (out): 5'-CAGCTCTGCCTTCGACACCGAA-3' (SEQ ID NO: 100) CG60-H (in): 5'-ATCACCAAGGATGGTGTCACCGT-3' (SEQ ID NO: 2)

[0250] Sequencing of the DNA fragment amplified using these primers revealed the complete sequence of the 5'-end of the Candida glabrata Hsp60 gene. The sequencing strategy is illustrated in FIG. 20, and the complete DNA sequence of the Candida glabrata Hsp60 gene and of its predicted translation product are provided in FIG. 21.

Example 10

Candida Glabrata Hsp60 Expression Plasmids

[0251] An internal 1.6 kbp-long fragment of the Candida glabrata Hsp60 gene cloned in vector pCR2.1 (see before and in FIG. 20) was PCR-amplified using primers CG60-Z and CG60-MA: TABLE-US-00029 CG60-MA: 5'-GATATACATATGGCCAAGGAGTTGAAG-3' (SEQ ID NO: 14)

[0252] PCR amplification with these primers and insertion of the amplified DNA into pET24A+adds a methionine and an alanine upstream from the first codon encoded by the 1.6 kbp fragment of the Candida glabrata Hsp60 gene. Thus, the Candida glabrata Hsp60 expression construct (in vector pET24A+) encodes a protein starting with the sequence MAKELK, which closely resembles typical bacterial Hsp60 N-terminus. The C-terminus of the encoded protein corresponds to the natural Candida glabrata sequence and contains PGM repeats (see FIGS. 23 and 25).

[0253] The above PCR fragment was not only cloned into the pET24A+ but also into the pET28A+T7 expression vector. Recombinant plasmids were identified by restriction analysis and confirmed by DNA sequencing.

[0254] FIGS. 22 and 23 provide the restriction map and relevant nucleotide sequence of the pET24A+-derived expression plasmid pETCG60A, and FIGS. 24 and 25 of the pET28A+-derived expression plasmid pETCG60AH.

Example 11

Expression and Purification of Recombinant Candida Glabrata Hsp60

[0255] E. coli BL21 (DE3) was transformed with pETCG60A and pETCG60AH, and expression of recombinant protein was monitored after induction with IPTG. Data presented in FIG. 26 demonstrate low-level, induced expression of a recombinant protein of approximately 60 kDa (indicated by arrowhead) in cells transformed with pETCG60 and of a slightly larger protein in cells transformed with pETCG60H.

[0256] Despite low expression level, His-tagged recombinant Candida glabrata Hsp60 could be effectively purified on a Ni-Sepharose column as illustrated in FIG. 27. The purification protocol used was similar to that described under Example 4.

[0257] From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims. Sequence CWU 1

100 1 2465 DNA Nesseria meningitidis 1 caattcaaca tactgaacgc caaagatatg gatgcagaac aagtctccct ttccaaagaa 60 tgcgacatca tcgagtcttc acacgactgg gaaaaagagt acggcaactt gaacgaacag 120 gaaatgctcg ccggcatcgt ctatgaataa acctgcctgc catttgaaac attatgcttg 180 aatgcattgg agccaaatgt attaaatcaa atataaaacc aatatattca taaagttata 240 tacttatagc catgctgtag cttgaaacag ttccaacata cccgccgccc gccctattta 300 cagcccatcg ggacaaaatg tttctcaaaa taagcaaaat caaataggat tatccacatg 360 gcaaaagtaa tcggtatcga cttaggtaca accaactctt gtttggccat ttccgaaaac 420 ggtcaaacca aagtgatcga aaacgcagaa ggcgcacgca ccacgccgtc cgttatcgct 480 tatttggacg gcggcgaaat cctcgtcggt gcgcctgcca aacgccaagc ggtaaccaac 540 gccaaaaaca ccatttacgc cgccaaacgt ttgatcggcc acaaatttga agacaaagaa 600 gtccaacgcg acatcgaatc tatgcctttc gaaatcatca aagccaacaa cggcgacgca 660 tgggtaaaag cacaaggcaa agagctgtct cctcctcaaa tttccgcaga agtcctgcgt 720 aaaatgaaag aagccgccga agcttacttg ggcgaaaaag taaccgaagc cgtgattacc 780 gtccctgcct acttcaacga cagccaacgt caagccacca aagacgcagg ccgtatcgcc 840 ggtttggacg tgaaacgcat catcaacgag ccgaccgcag ccgctttggc attcggtatg 900 gacaaaggcg acaacaaaga ccgcaaagta gccgtatatg acttgggcgg cggtactttc 960 gatatttcca tcatcgaaat cgccaacctc gacggcgaca aacaattcga agtattggca 1020 accaacggcg ataccttctt gggcggtgaa gacttcgacc aacgcctcat cgaccacatc 1080 atcgccgagt tcaaaaaaga acaaggcatt gatttgaaac aagacgtgat ggctctacaa 1140 cgcctgaaag aagctgccga aaaagccaaa atcgaattgt ccagcggcca gcaaaccgaa 1200 attaacctgc cgtacatcac catggacgca accggcccga aacacttggc gatgaaaatt 1260 acccgcgcca aattcgaaag cctggttgaa gacctgatta cccgctctat cgaaccttgc 1320 aaaattgcat tgaaagatgc cggcttgagc accggcgaca tcgacgacgt aatcttggtc 1380 ggcgggcagt cccgtatgcc gaaagtacaa gaagccgtta aagccttctt cggcaaagaa 1440 ccgcgcaaag acgtgaaccc tgacgaagcc gttgccgtag gcgcagcgat ccaaggcgaa 1500 gtattgagcg gcggccgcag cgacgtattg ctactggacg taactcctct gtctttgggt 1560 atcgaaacca tgggcggcgt gatgaccaaa ctgattcaga agaacaccac catcccgacc 1620 aaagcgtcgc aagtgttctc taccgccgaa gacaaccaaa gcgcagtaac catccacgta 1680 ctgcaaggcg aacgcgaacg cgcttctgcc aacaaatctt tgggtcagtt caacttgggc 1740 gacatcgcac ctgcaccgcg cggtatgccg caaatcgaag taaccttcga catcgacgcc 1800 aacggcatcc tgcacgtttc cgccaaagac aaaggcaccg gtaaagcagc caacatcacc 1860 atccaaggtt cttcaggttt gggcgaagaa gaaatcgaac gcatggtgaa agatgccgaa 1920 gccaatgccg aggaagataa aaaactgact gaattggtcg cttcccgcaa ccaagccgaa 1980 gccctgattc actctgtgaa gaaatctttg gccgactacg gcgacaaact cgatgcagcc 2040 gagaaagaaa aaattgaagc cgcgctgaaa gaagccgaag aagcagttaa aggcgacgac 2100 aaagccgcta tcgatgccaa aaccgaggcg ctgggcgcag ccagccaaaa actgggggaa 2160 atggtttacg ctcaagcaca agctgaagcc caagcaggcg aaagcgaaca agccaatgct 2220 tctgcaaaga aagacgatga tgtcgtagat gccgactttg aagaagtaaa agacgacaaa 2280 aaataattaa taccgtctga aaaaagcgcg aaccgtttga ttcgcgcttt tttcaattga 2340 gataaaagac catagcataa cagaggttcc aagttcatct accgtaatat tcccaaaccc 2400 aggttccaac tgttgtatcg gttctctgga attttttata tagtggatta aatttaaacc 2460 agtac 2465 2 23 DNA Artificial Sequence Primer Used to clone Candida glabrata Hsp60 gene and to contruct Candida glabrata Hsp60 expression plasmids 2 atcaccaagg atggtgtcac cgt 23 3 1929 DNA Nesseria meningitidis 3 atggcaaaag taatcggtat cgacttaggt acaaccaact cttgtttggc catttccgaa 60 aacggtcaaa ccaaagtgat cgaaaacgca gaaggcgcac gcaccacgcc gtccgttatc 120 gcttatttgg acggcggcga aatcctcgtc ggtgcgcctg ccaaacgcca agcggtaacc 180 aacgccaaaa acaccattta cgccgccaaa cgtttgatcg gccacaaatt tgaagacaaa 240 gaagtccaac gcgacatcga atctatgcct ttcgaaatca tcaaagccaa caacggcgac 300 gcatgggtaa aagcacaagg caaagagctg tctcctcctc aaatttccgc agaagtcctg 360 cgtaaaatga aagaagccgc cgaagcttac ttgggcgaaa aagtaaccga agccgtgatt 420 accgtccctg cctacttcaa cgacagccaa cgtcaagcca ccaaagacgc aggccgtatc 480 gccggtttgg acgtgaaacg catcatcaac gagccgaccg cagccgcttt ggcattcggt 540 atggacaaag gcgacaacaa agaccgcaaa gtagccgtat atgacttggg cggcggtact 600 ttcgatattt ccatcatcga aatcgccaac ctcgacggcg acaaacaatt cgaagtattg 660 gcaaccaacg gcgatacctt cttgggcggt gaagacttcg accaacgcct catcgaccac 720 atcatcgccg agttcaaaaa agaacaaggc attgatttga aacaagacgt gatggctcta 780 caacgcctga aagaagctgc cgaaaaagcc aaaatcgaat tgtccagcgg ccagcaaacc 840 gaaattaacc tgccgtacat caccatggac gcaaccggcc cgaaacactt ggcgatgaaa 900 attacccgcg ccaaattcga aagcctggtt gaagacctga ttacccgctc tatcgaacct 960 tgcaaaattg cattgaaaga tgccggcttg agcaccggcg acatcgacga cgtaatcttg 1020 gtcggcgggc agtcccgtat gccgaaagta caagaagccg ttaaagcctt cttcggcaaa 1080 gaaccgcgca aagacgtgaa ccctgacgaa gccgttgccg taggcgcagc gatccaaggc 1140 gaagtattga gcggcggccg cagcgacgta ttgctactgg acgtaactcc tctgtctttg 1200 ggtatcgaaa ccatgggcgg cgtgatgacc aaactgattc agaagaacac caccatcccg 1260 accaaagcgt cgcaagtgtt ctctaccgcc gaagacaacc aaagcgcagt aaccatccac 1320 gtactgcaag gcgaacgcga acgcgcttct gccaacaaat ctttgggtca gttcaacttg 1380 ggcgacatcg cacctgcacc gcgcggtatg ccgcaaatcg aagtaacctt cgacatcgac 1440 gccaacggca tcctgcacgt ttccgccaaa gacaaaggca ccggtaaagc agccaacatc 1500 accatccaag gttcttcagg tttgagcgaa gaagaaatcg aacgcatggt gaaagatgcc 1560 gaagccaatg ccgaggaaga taaaaaactg actgaattgg tcgcttcccg caaccaagcc 1620 gaagccctga ttcactctgt gaaaaaatct ttggccgact acggcgacaa actcgatgca 1680 gccgagaaag aaaaaattga agccgcgctg aaagaagccg aagaagcagt taaaggcgac 1740 gacaaagccg ctatcgatgc caaaaccgag gcgctgggcg cagccagcca aaaactgggg 1800 gaaatggttt acgctcaagc acaagctgaa gcccaagcag gcgaaagcga acaagccaat 1860 gcttctgcaa agaaagacga tgatgtcgta gatgccgact ttgaagaagt aaaagacgac 1920 aaaaaataa 1929 4 1989 DNA Nesseria meningitidis 4 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggcaaaag taatcggtat cgacttaggt acaaccaact cttgtttggc catttccgaa 120 aacggtcaaa ccaaagtgat cgaaaacgca gaaggcgcac gcaccacgcc gtccgttatc 180 gcttatttgg acggcggcga aatcctcgtc ggtgcgcctg ccaaacgcca agcggtaacc 240 aacgccaaaa acaccattta cgccgccaaa cgtttgatcg gccacaaatt tgaagacaaa 300 gaagtccaac gcgacatcga atctatgcct ttcgaaatca tcaaagccaa caacggcgac 360 gcatgggtaa aagcacaagg caaagagctg tctcctcctc aaatttccgc agaagtcctg 420 cgtaaaatga aagaagccgc cgaagcttac ttgggcgaaa aagtaaccga agccgtgatt 480 accgtccctg cctacttcaa cgacagccaa cgtcaagcca ccaaagacgc aggccgtatc 540 gccggtttgg acgtgaaacg catcatcaac gagccgaccg cagccgcttt ggcattcggt 600 atggacaaag gcgacaacaa agaccgcaaa gtagccgtat atgacttggg cggcggtact 660 ttcgatattt ccatcatcga aatcgccaac ctcgacggcg acaaacaatt cgaagtattg 720 gcaaccaacg gcgatacctt cttgggcggt gaagacttcg accaacgcct catcgaccac 780 atcatcgccg agttcaaaaa agaacaaggc attgatttga aacaagacgt gatggctcta 840 caacgcctga aagaagctgc cgaaaaagcc aaaatcgaat tgtccagcgg ccagcaaacc 900 gaaattaacc tgccgtacat caccatggac gcaaccggcc cgaaacactt ggcgatgaaa 960 attacccgcg ccaaattcga aagcctggtt gaagacctga ttacccgctc tatcgaacct 1020 tgcaaaattg cattgaaaga tgccggcttg agcaccggcg acatcgacga cgtaatcttg 1080 gtcggcgggc agtcccgtat gccgaaagta caagaagccg ttaaagcctt cttcggcaaa 1140 gaaccgcgca aagacgtgaa ccctgacgaa gccgttgccg taggcgcagc gatccaaggc 1200 gaagtattga gcggcggccg cagcgacgta ttgctactgg acgtaactcc tctgtctttg 1260 ggtatcgaaa ccatgggcgg cgtgatgacc aaactgattc agaagaacac caccatcccg 1320 accaaagcgt cgcaagtgtt ctctaccgcc gaagacaacc aaagcgcagt aaccatccac 1380 gtactgcaag gcgaacgcga acgcgcttct gccaacaaat ctttgggtca gttcaacttg 1440 ggcgacatcg cacctgcacc gcgcggtatg ccgcaaatcg aagtaacctt cgacatcgac 1500 gccaacggca tcctgcacgt ttccgccaaa gacaaaggca ccggtaaagc agccaacatc 1560 accatccaag gttcttcagg tttgagcgaa gaagaaatcg aacgcatggt gaaagatgcc 1620 gaagccaatg ccgaggaaga taaaaaactg actgaattgg tcgcttcccg caaccaagcc 1680 gaagccctga ttcactctgt gaaaaaatct ttggccgact acggcgacaa actcgatgca 1740 gccgagaaag aaaaaattga agccgcgctg aaagaagccg aagaagcagt taaaggcgac 1800 gacaaagccg ctatcgatgc caaaaccgag gcgctgggcg cagccagcca aaaactgggg 1860 gaaatggttt acgctcaagc acaagctgaa gcccaagcag gcgaaagcga acaagccaat 1920 gcttctgcaa agaaagacga tgatgtcgta gatgccgact ttgaagaagt aaaagacgac 1980 aaaaaataa 1989 5 2480 DNA Aspergillus fumigatus 5 gtacgaattt ccccttccga cgatccgaga acgtccctcg ggaaggccac acgtgacctt 60 ctaggagctt ctcccgccaa gacatccggg gatcgagaat cgcctggaaa aatttcgaga 120 ctttggcttc atctccccag ctttcatctc cattccatct tccttacctt ctattccccc 180 tcttctcttc cttctctgca cctgttcttg ctctgggagg ttcgatcggg cagtagtgtt 240 catcttaacg ttgattatat tctcttctat cccgtccttt catcaccctt ctttccataa 300 tgcagagagc tctttcttcc aggacgtctg tcctttccgc tgcctccaaa cgggctgctt 360 tcaccaagcc cgctggcctt aacctgcagc agcagcgttt cgcccacaag gtatgttttc 420 atctacaatc tagaatttta agcttctgaa gtggtgccaa tttctccgtg tcacccggag 480 ctcaaccccg ataccttgct aacgaacttt caggagctca agttcggtgt cgaagcccgt 540 gctcagctcc tcaagggtgt tgacactctg gccaaggccg tgacttcgac tcttggtcct 600 aagggtcgta acgtccttat cgagtctccc tatggctccc ctaagatcac caagggtacg 660 tttgactcga gttaacccaa gtcgctgctt tcacaaacga attgtggttc tgactaaaaa 720 tagatggtgt ctctgttgcc aaggccatca ctctccaaga caagttcgag aacctcggtg 780 ctcgcctcct ccaggatgtc gcttctaaga ccaacgagat tgctggtgac ggtaccacca 840 ccgctaccgt ccttgcccgt gccatcttct ctgagaccgt gaagaatgtt gctgctggct 900 gcaaccccat ggatctgcgc cgcggtatcc aggctgctgt tgatgctgtc gtcgactacc 960 tccagaagaa caagcgtgac atcaccaccg gtgaggagat cgctcaggtt gctactatct 1020 ccgctaacgg tgacacccac attggtaagc tgatctccac cgccatggag cgtgttggca 1080 aggagggtgt catcactgtc aaggagggca agaccattga ggatgagctc gaggtcactg 1140 agggtatgcg cttcgaccgt ggatacacct ccccctactt catcaccgat accaagtccc 1200 agaaggttga gttcgagaag cctctgattc tgctgtctga gaagaagatc tctgccgttc 1260 aggacatcat ccccgccctt gaggcctcca ccaccctccg ccgccccctg gttattatcg 1320 cagaggacat tgagggtgag gctctcgccg tctgcattct gaacaagctt cgtggccagc 1380 tgcaggtcgc tgctgtcaag gctcctggat tcggtgacaa ccgcaagagc atcctgggcg 1440 atcttgccgt ccttaccaac ggtaccgtct tcactgatga gctcgacatc aaactcgaga 1500 agcttacccc cgatatgctt ggttccaccg gcgccatcac catcaccaag gaggacacca 1560 tcatcctgaa cggggagggc agcaaggacg ccattgccca gcgctgcgag cagattcgcg 1620 gtgtcatggc ggaccccagc acctccgaat acgagaagga gaagctccag gagcgtctag 1680 ctaagctctc tggcggtgtt gccgtcatca aggtcggtgg tgcctccgag gttgaggtcg 1740 gtgagaagaa ggaccgtgtt gtcgatgctc tcaatgctac ccgtgctgct gttgaggagg 1800 gtatcctccc cggtggtggt accgcccttc tcaaggccgc cgccaacggc cttgacaatg 1860 tcaagcccga gaacttcgac cagcaactcg gtgtgagcat catcaagaat gccatcaccc 1920 gccccgctcg caccattgtt gagaacgccg gcctcgaggg cagcgtcatt gtcggcaagc 1980 tgaccgacga gttcgccaag gacttcaacc gcggtttcga cagctccaag ggcgagtacg 2040 tcgacatgat ctccagcggt atcctcgatc ccctcaaggt tgttcgcacc gctctgctcg 2100 acgccagcgg tgtcgcctcc ctgctcggta ccactgaggt cgctattgtt gaggcccctg 2160 aggagaaggg ccccgctgct cctggcatgg gtggtatggg tggtatgggc ggcatgggtg 2220 gcggcatgtt ctaagctgct cccagttgcc tttgctacca tagcctcttc catgatttaa 2280 aggtttaact tccctttcga gcgtgtcttt gcatgtacga gcatttcctg atatatcggt 2340 gttgagagtt ttctgtaatt tttcctttgt ttctgatgtg ttacacgcct tgacagcccc 2400 ttcacctact ccgacttcgt cttatacctc gatactcata tctccctctt cgacccgcct 2460 cccctttgat tgactcgatc 2480 6 1653 DNA Aspergillus fumigatus 6 atgaaagagc tcaagttcgg tgtcgaagcc cgtgctcagc tcctcaaggg tgttgacact 60 ctggccaagg ccgtgacttc gactcttggt cctaagggtc gtaacgtcct tatcgagtct 120 ccctatggct cccctaagat caccaaggat ggtgtctctg ttgccaaggc catcactctc 180 caagacaagt tcgagaacct cggtgctcgc ctcctccagg atgtcgcttc taagaccaac 240 gagattgctg gtgacggtac caccaccgct accgtccttg cccgtgccat cttctctgag 300 accgtgaaga atgttgctgc tggctgcaac cccatggatc tgcgccgcgg tatccaggct 360 gctgttgatg ctgtcgtcga ctacctccag aagaacaagc gtgacatcac caccggtgag 420 gagatcgctc aggttgctac tatctccgct aacggtgaca cccacattgg taagctgatc 480 tccaccgcca tggagcgtgt tggcaaggag ggtgtcatca ctgtcaagga gggcaagacc 540 attgaggatg agctcgaggt cactgagggt atgcgcttcg accgtggata cacctccccc 600 tacttcatca ccgataccaa gtcccagaag gttgagttcg agaagcctct gattctgctg 660 tctgagaaga agatctctgc cgttcaggac atcatccccg cccttgaggc ctccaccacc 720 ctccgccgcc ccctggttat tatcgcagag gacattgagg gtgaggctct cgccgtctgc 780 attctgaaca agcttcgtgg ccagctgcag gtcgctgctg tcaaggctcc tggattcggt 840 gacaaccgca agagcatcct gggcgatctt gccgtcctta ccaacggtac cgtcttcact 900 gatgagctcg acatcaaact cgagaagctt acccccgata tgcttggttc caccggcgcc 960 atcaccatca ccaaggagga caccatcatc ctgaacgggg agggcagcaa ggacgccatt 1020 gcccagcgct gcgagcagat tcgcggtgtc atggcggacc ccagcacctc cgaatacgag 1080 aaggagaagc tccaggagcg tctagctaag ctctctggcg gtgttgccgt catcaaggtc 1140 ggtggtgcct ccgaggttga ggtcggtgag aagaaggacc gtgttgtcga tgctctcaat 1200 gctacccgtg ctgctgttga ggagggtatc ctccccggtg gtggtaccgc ccttctcaag 1260 gccgccgcca acggccttga caatgtcaag cccgagaact tcgaccagca actcggtgtg 1320 agcatcatca agaatgccat cacccgcccc gctcgcacca ttgttgagaa cgccggcctc 1380 gagggcagcg tcattgtcgg caagctgacc gacgagttcg ccaaggactt caaccgcggt 1440 ttcgacagct ccaagggcga gtacgtcgac atgatctcca gcggtatcct cgatcccctc 1500 aaggttgttc gcaccgctct gctcgacgcc agcggtgtcg cctccctgct cggtaccact 1560 gaggtcgcta ttgttgaggc ccctgaggag aagggccccg ctgctcctgg catgggtggt 1620 atgggtggta tgggcggcat gggcggcatg tag 1653 7 1713 DNA Aspergillus fumigatus 7 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atgaaagagc tcaagttcgg tgtcgaagcc cgtgctcagc tcctcaaggg tgttgacact 120 ctggccaagg ccgtgacttc gactcttggt cctaagggtc gtaacgtcct tatcgagtct 180 ccctatggct cccctaagat caccaaggat ggtgtctctg ttgccaaggc catcactctc 240 caagacaagt tcgagaacct cggtgctcgc ctcctccagg atgtcgcttc taagaccaac 300 gagattgctg gtgacggtac caccaccgct accgtccttg cccgtgccat cttctctgag 360 accgtgaaga atgttgctgc tggctgcaac cccatggatc tgcgccgcgg tatccaggct 420 gctgttgatg ctgtcgtcga ctacctccag aagaacaagc gtgacatcac caccggtgag 480 gagatcgctc aggttgctac tatctccgct aacggtgaca cccacattgg taagctgatc 540 tccaccgcca tggagcgtgt tggcaaggag ggtgtcatca ctgtcaagga gggcaagacc 600 attgaggatg agctcgaggt cactgagggt atgcgcttcg accgtggata cacctccccc 660 tacttcatca ccgataccaa gtcccagaag gttgagttcg agaagcctct gattctgctg 720 tctgagaaga agatctctgc cgttcaggac atcatccccg cccttgaggc ctccaccacc 780 ctccgccgcc ccctggttat tatcgcagag gacattgagg gtgaggctct cgccgtctgc 840 attctgaaca agcttcgtgg ccagctgcag gtcgctgctg tcaaggctcc tggattcggt 900 gacaaccgca agagcatcct gggcgatctt gccgtcctta ccaacggtac cgtcttcact 960 gatgagctcg acatcaaact cgagaagctt acccccgata tgcttggttc caccggcgcc 1020 atcaccatca ccaaggagga caccatcatc ctgaacgggg agggcagcaa ggacgccatt 1080 gcccagcgct gcgagcagat tcgcggtgtc atggcggacc ccagcacctc cgaatacgag 1140 aaggagaagc tccaggagcg tctagctaag ctctctggcg gtgttgccgt catcaaggtc 1200 ggtggtgcct ccgaggttga ggtcggtgag aagaaggacc gtgttgtcga tgctctcaat 1260 gctacccgtg ctgctgttga ggagggtatc ctccccggtg gtggtaccgc ccttctcaag 1320 gccgccgcca acggccttga caatgtcaag cccgagaact tcgaccagca actcggtgtg 1380 agcatcatca agaatgccat cacccgcccc gctcgcacca ttgttgagaa cgccggcctc 1440 gagggcagcg tcattgtcgg caagctgacc gacgagttcg ccaaggactt caaccgcggt 1500 ttcgacagct ccaagggcga gtacgtcgac atgatctcca gcggtatcct cgatcccctc 1560 aaggttgttc gcaccgctct gctcgacgcc agcggtgtcg cctccctgct cggtaccact 1620 gaggtcgcta ttgttgaggc ccctgaggag aagggccccg ctgctcctgg catgggtggt 1680 atgggtggta tgggcggcat gggcggcatg tag 1713 8 2051 DNA Candida glabrata 8 ccgggtaaag tacctgattg cgcacttaca gctaacagct gacgcactcg agaaatctgc 60 ccgttttgtt catggaaact tgaagaaaat cagggaaatc gttactgcgc tctctctaac 120 gcttgcaagc tcctggaata caaattcgca aagtatataa ctctatagct ttcaaccttg 180 ttactgtgga gtagctgtta agggatagag acataagata aaccatacca tacataaatc 240 accccccata taaacaaatg ttgagagctg ttgcacgttc gcaggttaga tctttgagaa 300 acgctcgttt gtactccagt ttcaaggagt tgaagttcgg tgtcgaaggc agagctgctc 360 tgcttcgtgg tgtcgagact ttggccgacg ctgtctctgc cacgctgggg cctaagggta 420 gaaatgtgct gatcgagcag ccattcggag caccaaagat caccaaggat ggtgtcaccg 480 tggccagatc cattactttg gaggacaagt tcgagaacat gggtgctaag cttctgcaag 540 aagttgcctc caagactaac gaggccgccg gtgacggtac cacctccgcc actgtcttgg 600 gtagagccat cttcaccgag tccgtcaaga acgtcgctgc cggttgcaac cctatggatt 660 tgagaagggg ttcccaggcc gccgtcgaga aggtcatcca attcttgact gaaaacaaga 720 aggagatcac cacttctgag gaaatcgccc aggtggccac catctcagct aacggtgacg 780 ctcacgtcgg taagttgctt gcctccgcca tggaaaaggt tggcaaggaa ggtgttatca 840 ccatcagaga aggcagaact ttggaagacg aactagaagt caccgaaggt atgagatttg 900 accgtggttt catctcccca tacttcatca ctgacgcaaa gtccggcaag gtagaattcg 960 aaaagccatt gttgttgttg agcgaaaaga agatctcttc catccaagac atcttgccag 1020 ctttggaact atctaaccaa agtagaagac cactattgat catcgccgaa gatgtcgacg 1080 gtgaagccct agctgcttgt atcctaaaca agttgagagg ccaagtcaag gtctgtgccg 1140 ttaaggctcc aggtttcggt gacaacagaa agaacattct aggtgatgtc gccatcttga 1200 ccggcagtac tgtttttact gaagaattgg acttgaagcc agaacaagcc actatggaac 1260 acctaggttc ctgtgactcc attactatca caaaggaaga cactgttatc ctaaacggta 1320 acggctccaa ggactctatc caagaaagaa ttgaacagat caagaactcc attgatgtca 1380 ccactactaa ctcttacgag aaggagaagc tacaagaaag acttgccaag ttatccggtg 1440 gtgttgctgt catcagggtt ggtggtgctt ctgaagttga agtcggtgaa aagaaggacc 1500 gttacgatga cgctttgaat gccaccagag ctgccgttga agaaggtatc ttaccaggtg 1560 gtggtactgc tttggttaag gcctctagag ttttagacga agtcaagact gagaacttcg 1620 accaaaaatt gggagttgac attatcagaa aggccattac tagaccagct aagcaaatta 1680 ttgagaacgc cggtgaagaa ggctccgtta ttgtcggtaa gcttgttgat gaatttggcg 1740 aagattttgc taagggttac gactccgcta agggagaatt cactgatatg ttggctgccg 1800 gtattattga cccattcaaa gtcgttagat ctggtctggt cgacgcttcc ggtgttgctt 1860 ccttgttggc tactaccgaa gttgccatcg ttgacgctcc tgaaccagct ccagctgctg 1920 gtgccccagg tggtggtatg ccaggtatgc caggtatgat gtaaaaggtc taacttttgc 1980 aatcatgctg gtgaaaatga agcaaatcat tacatagagt ggtaaaatct tcaagaccaa 2040 atagcttgta c 2051 9 1647 DNA Candida glabrata 9 atggccaagg agttgaagtt tggggtcgaa

ggcagagctg ctctgcttcg tggtgtcgag 60 actttggccg acgctgtctc tgccacgctg gggcctaagg gtagaaatgt gctgatcgag 120 cagccattcg gagcaccaaa gatcaccaag gatggtgtca ccgtggccag atccattact 180 ttggaggaca agttcgagaa catgggtgct aagcttctgc aagaagttgc ctccaagact 240 aacgaggccg ccggtgacgg taccacctcc gccactgtct tgggtagagc catcttcacc 300 gagtccgtca agaacgtcgc tgccggttgc aaccctatgg atttgagaag gggttcccag 360 gccgccgtcg agaaggtcat ccaattcttg actgaaaaca agaaggagat caccacttct 420 gaggaaatcg cccaggtggc caccatctca gctaacggtg acgctcacgt cggtaagttg 480 cttgcctccg ccatggaaaa ggttggcaag gaaggtgtta tcaccatcag agaaggcaga 540 actttggaag acgaactaga agtcaccgaa ggtatgagat ttgaccgtgg tttcatctcc 600 ccatacttca tcactgacgc aaagtccggc aaggtagaat tcgaaaagcc attgttgttg 660 ttgagcgaaa agaagatctc ttccatccaa gacatcttgc cagctttgga actatctaac 720 caaagtagaa gaccactatt gatcatcgcc gaagatgtcg acggtgaagc cctagctgct 780 tgtatcctaa acaagttgag aggccaagtc aaggtctgtg ccgttaaggc tccaggtttc 840 ggtgacaaca gaaagaacat tctaggtgat gtcgccatct tgaccggcag tactgttttt 900 actgaagaat tggacttgaa gccagaacaa gccactatgg aacacctagg ttcctgtgac 960 tccattacta tcacaaagga agacactgtt atcctaaacg gtaacggctc caaggactct 1020 atccaagaaa gaattgaaca gatcaagaac tccattgatg tcaccactac taactcttac 1080 gagaaggaga agctacaaga aagacttgcc aagttatccg gtggtgttgc tgtcatcagg 1140 gttggtggtg cttctgaagt tgaagtcggt gaaaagaagg accgttacga tgacgctttg 1200 aatgccacca gagctgccgt tgaagaaggt atcttaccag gtggtggtac tgctttggtt 1260 aaggcctcta gagttttaga cgaagtcaag actgagaact tcgaccaaaa attgggagtt 1320 gacattatca gaaaggccat tactagacca gctaagcaaa ttattgagaa cgccggtgaa 1380 gaaggctccg ttattgtcgg taagcttgtt gatgaatttg gcgaagattt tgctaagggt 1440 tacgactccg ctaagggaga attcactgat atgttggctg ccggtattat tgacccattc 1500 aaagtcgtta gatctggtct ggtcgacgct tccggtgttg cttccttgtt ggctactacc 1560 gaagttgcca tcgttgacgc tcctgaacca gctccagctg ctggtgcccc aggtggtggt 1620 atgccaggta tgccaggtat gatgtaa 1647 10 1707 DNA Candida glabrata 10 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggccaagg agttgaagtt tggggtcgaa ggcagagctg ctctgcttcg tggtgtcgag 120 actttggccg acgctgtctc tgccacgctg gggcctaagg gtagaaatgt gctgatcgag 180 cagccattcg gagcaccaaa gatcaccaag gatggtgtca ccgtggccag atccattact 240 ttggaggaca agttcgagaa catgggtgct aagcttctgc aagaagttgc ctccaagact 300 aacgaggccg ccggtgacgg taccacctcc gccactgtct tgggtagagc catcttcacc 360 gagtccgtca agaacgtcgc tgccggttgc aaccctatgg atttgagaag gggttcccag 420 gccgccgtcg agaaggtcat ccaattcttg actgaaaaca agaaggagat caccacttct 480 gaggaaatcg cccaggtggc caccatctca gctaacggtg acgctcacgt cggtaagttg 540 cttgcctccg ccatggaaaa ggttggcaag gaaggtgtta tcaccatcag agaaggcaga 600 actttggaag acgaactaga agtcaccgaa ggtatgagat ttgaccgtgg tttcatctcc 660 ccatacttca tcactgacgc aaagtccggc aaggtagaat tcgaaaagcc attgttgttg 720 ttgagcgaaa agaagatctc ttccatccaa gacatcttgc cagctttgga actatctaac 780 caaagtagaa gaccactatt gatcatcgcc gaagatgtcg acggtgaagc cctagctgct 840 tgtatcctaa acaagttgag aggccaagtc aaggtctgtg ccgttaaggc tccaggtttc 900 ggtgacaaca gaaagaacat tctaggtgat gtcgccatct tgaccggcag tactgttttt 960 actgaagaat tggacttgaa gccagaacaa gccactatgg aacacctagg ttcctgtgac 1020 tccattacta tcacaaagga agacactgtt atcctaaacg gtaacggctc caaggactct 1080 atccaagaaa gaattgaaca gatcaagaac tccattgatg tcaccactac taactcttac 1140 gagaaggaga agctacaaga aagacttgcc aagttatccg gtggtgttgc tgtcatcagg 1200 gttggtggtg cttctgaagt tgaagtcggt gaaaagaagg accgttacga tgacgctttg 1260 aatgccacca gagctgccgt tgaagaaggt atcttaccag gtggtggtac tgctttggtt 1320 aaggcctcta gagttttaga cgaagtcaag actgagaact tcgaccaaaa attgggagtt 1380 gacattatca gaaaggccat tactagacca gctaagcaaa ttattgagaa cgccggtgaa 1440 gaaggctccg ttattgtcgg taagcttgtt gatgaatttg gcgaagattt tgctaagggt 1500 tacgactccg ctaagggaga attcactgat atgttggctg ccggtattat tgacccattc 1560 aaagtcgtta gatctggtct ggtcgacgct tccggtgttg cttccttgtt ggctactacc 1620 gaagttgcca tcgttgacgc tcctgaacca gctccagctg ctggtgcccc aggtggtggt 1680 atgccaggta tgccaggtat gatgtaa 1707 11 1005 DNA Neisseria meningitidis 11 cctgcdtatt tcaacgacag ccaacgtcaa gccaccaaag acgcaggccg tatcgccggt 60 ttggacgtga aacgcatcat caacgagccg accgcagccg ctttggcatt cggtatggac 120 aaaggcgaca acaaagaccg caaagtagcc gtatatgact tgggcggcgg tactttcgat 180 atttccatca tcgaaatcgc caacctcgac ggcgacaaac aattcgaagt attggcaacc 240 aacggcgata ccttcttggg cggtgaagac ttcgaccaac gcctcatcga ccacatcatc 300 gccgagttca aaaaagaaca aggcattgat ttgaaacaag acgtgatggc tctacaacgc 360 ctgaaagaag ctgccgaaaa agccaaaatc gaattgtcca gcggccagca aaccgaaatt 420 aacctgccgt acatcaccat ggacgcaacc ggcccgaaac acttggcgat gaaaattacc 480 cgcgccaaat tcgaaagcct ggttgaagac ctgattaccc gctctatcga accttgcaaa 540 attgcattga aagatgccgg cttgagcacc ggcgacatcg acgacgtaat cttggtcggc 600 gggcagtccc gtatgccgaa agtacaagaa gccgttaaag ccttcttcgg caaagaaccg 660 cgcaaagacg tgaaccctga cgaagccgtt gccgtaggcg cagcgatcca aggcgaagta 720 ttgagcggcg gccgcagcga cgtattgcta ctggacgtaa ctcctctgtc tttgggtatc 780 gaaaccatgg gcggcgtgat gaccaaactg attcagaaga acaccaccat cccgaccaaa 840 gcgtcgcaag tgttctctac cgccgaagac aaccaaagcg cagtaaccat ccacgtactg 900 caaggcgaac gcgaacgcgc ttctgccaac aaatctttgg gtcagttcaa cttgggcgac 960 atcgcacctg caccgcgcgg tatgccacaa atcgaagtaa chttt 1005 12 415 PRT Neisseria meningitidis 12 Met Ala Lys Val Ile Gly Ile Asp Leu Gly Thr Thr Asn Ser Cys Leu 1 5 10 15 Ala Ile Ser Glu Asn Gly Gln Thr Lys Val Ile Glu Asn Ala Glu Gly 20 25 30 Ala Arg Thr Thr Pro Ser Val Ile Ala Tyr Leu Asp Gly Gly Glu Ile 35 40 45 Leu Val Gly Ala Pro Ala Lys Arg Gln Ala Val Thr Asn Ala Lys Asn 50 55 60 Thr Ile Tyr Ala Ala Lys Arg Leu Ile Gly His Lys Phe Glu Asp Lys 65 70 75 80 Pro Ala Tyr Phe Asn Asp Ser Gln Arg Gln Ala Thr Lys Asp Ala Gly 85 90 95 Arg Ile Ala Gly Leu Asp Val Lys Arg Ile Ile Asn Glu Pro Thr Ala 100 105 110 Ala Ala Leu Ala Phe Gly Met Asp Lys Gly Asp Asn Lys Asp Arg Lys 115 120 125 Val Ala Val Tyr Asp Leu Gly Gly Gly Thr Phe Asp Ile Ser Ile Ile 130 135 140 Glu Ile Ala Asn Leu Asp Gly Asp Lys Gln Phe Glu Val Leu Ala Thr 145 150 155 160 Asn Gly Asp Thr Phe Leu Gly Gly Glu Asp Phe Asp Gln Arg Leu Ile 165 170 175 Asp His Ile Ile Ala Glu Phe Lys Lys Glu Gln Gly Ile Asp Leu Lys 180 185 190 Gln Asp Val Met Ala Leu Gln Arg Leu Lys Glu Ala Ala Glu Lys Ala 195 200 205 Lys Ile Glu Leu Ser Ser Gly Gln Gln Thr Glu Ile Asn Leu Pro Tyr 210 215 220 Ile Thr Met Asp Ala Thr Gly Pro Lys His Leu Ala Met Lys Ile Thr 225 230 235 240 Arg Ala Lys Phe Glu Ser Leu Val Glu Asp Leu Ile Thr Arg Ser Ile 245 250 255 Glu Pro Cys Lys Ile Ala Leu Lys Asp Ala Gly Leu Ser Thr Gly Asp 260 265 270 Ile Asp Asp Val Ile Leu Val Gly Gly Gln Ser Arg Met Pro Lys Val 275 280 285 Gln Glu Ala Val Lys Ala Phe Phe Gly Lys Glu Pro Arg Lys Asp Val 290 295 300 Asn Pro Asp Glu Ala Val Ala Val Gly Ala Ala Ile Gln Gly Glu Val 305 310 315 320 Leu Ser Gly Gly Arg Ser Asp Val Leu Leu Leu Asp Val Thr Pro Leu 325 330 335 Ser Leu Gly Ile Glu Thr Met Gly Gly Val Met Thr Lys Leu Ile Gln 340 345 350 Lys Asn Thr Thr Ile Pro Thr Lys Ala Ser Gln Val Phe Ser Thr Ala 355 360 365 Glu Asp Asn Gln Ser Ala Val Thr Ile His Val Leu Gln Gly Glu Arg 370 375 380 Glu Arg Ala Ser Ala Asn Lys Ser Leu Gly Gln Phe Asn Leu Gly Asp 385 390 395 400 Ile Ala Pro Ala Pro Arg Gly Met Pro Gln Ile Glu Val Thr Phe 405 410 415 13 642 PRT Neisseria meningitidis 13 Met Ala Lys Val Ile Gly Ile Asp Leu Gly Thr Thr Asn Ser Cys Leu 1 5 10 15 Ala Ile Ser Glu Asn Gly Gln Thr Lys Val Ile Glu Asn Ala Glu Gly 20 25 30 Ala Arg Thr Thr Pro Ser Val Ile Ala Tyr Leu Asp Gly Gly Glu Ile 35 40 45 Leu Val Gly Ala Pro Ala Lys Arg Gln Ala Val Thr Asn Ala Lys Asn 50 55 60 Thr Ile Tyr Ala Ala Lys Arg Leu Ile Gly His Lys Phe Glu Asp Lys 65 70 75 80 Glu Val Gln Arg Asp Ile Glu Ser Met Pro Phe Glu Ile Ile Lys Ala 85 90 95 Asn Asn Gly Asp Ala Trp Val Lys Ala Gln Gly Lys Glu Leu Ser Pro 100 105 110 Pro Gln Ile Ser Ala Glu Val Leu Arg Lys Met Lys Glu Ala Ala Glu 115 120 125 Ala Tyr Leu Gly Glu Lys Val Thr Glu Ala Val Ile Thr Val Pro Ala 130 135 140 Tyr Phe Asn Asp Ser Gln Arg Gln Ala Thr Lys Asp Ala Gly Arg Ile 145 150 155 160 Ala Gly Leu Asp Val Lys Arg Ile Ile Asn Glu Pro Thr Ala Ala Ala 165 170 175 Leu Ala Phe Gly Met Asp Lys Gly Asp Asn Lys Asp Arg Lys Val Ala 180 185 190 Val Tyr Asp Leu Gly Gly Gly Thr Phe Asp Ile Ser Ile Ile Glu Ile 195 200 205 Ala Asn Leu Asp Gly Asp Lys Gln Phe Glu Val Leu Ala Thr Asn Gly 210 215 220 Asp Thr Phe Leu Gly Gly Glu Asp Phe Asp Gln Arg Leu Ile Asp His 225 230 235 240 Ile Ile Ala Glu Phe Lys Lys Glu Gln Gly Ile Asp Leu Lys Gln Asp 245 250 255 Val Met Ala Leu Gln Arg Leu Lys Glu Ala Ala Glu Lys Ala Lys Ile 260 265 270 Glu Leu Ser Ser Gly Gln Gln Thr Glu Ile Asn Leu Pro Tyr Ile Thr 275 280 285 Met Asp Ala Thr Gly Pro Lys His Leu Ala Met Lys Ile Thr Arg Ala 290 295 300 Lys Phe Glu Ser Leu Val Glu Asp Leu Ile Thr Arg Ser Ile Glu Pro 305 310 315 320 Cys Lys Ile Ala Leu Lys Asp Ala Gly Leu Ser Thr Gly Asp Ile Asp 325 330 335 Asp Val Ile Leu Val Gly Gly Gln Ser Arg Met Pro Lys Val Gln Glu 340 345 350 Ala Val Lys Ala Phe Phe Gly Lys Glu Pro Arg Lys Asp Val Asn Pro 355 360 365 Asp Glu Ala Val Ala Val Gly Ala Ala Ile Gln Gly Glu Val Leu Ser 370 375 380 Gly Gly Arg Ser Asp Val Leu Leu Leu Asp Val Thr Pro Leu Ser Leu 385 390 395 400 Gly Ile Glu Thr Met Gly Gly Val Met Thr Lys Leu Ile Gln Lys Asn 405 410 415 Thr Thr Ile Pro Thr Lys Ala Ser Gln Val Phe Ser Thr Ala Glu Asp 420 425 430 Asn Gln Ser Ala Val Thr Ile His Val Leu Gln Gly Glu Arg Glu Arg 435 440 445 Ala Ser Ala Asn Lys Ser Leu Gly Gln Phe Asn Leu Gly Asp Ile Ala 450 455 460 Pro Ala Pro Arg Gly Met Pro Gln Ile Glu Val Thr Phe Asp Ile Asp 465 470 475 480 Ala Asn Gly Ile Leu His Val Ser Ala Lys Asp Lys Gly Thr Gly Lys 485 490 495 Ala Ala Asn Ile Thr Ile Gln Gly Ser Ser Gly Leu Gly Glu Glu Glu 500 505 510 Ile Glu Arg Met Val Lys Asp Ala Glu Ala Asn Ala Glu Glu Asp Lys 515 520 525 Lys Leu Thr Glu Leu Val Ala Ser Arg Asn Gln Ala Glu Ala Leu Ile 530 535 540 His Ser Val Lys Lys Ser Leu Ala Asp Tyr Gly Asp Lys Leu Asp Ala 545 550 555 560 Ala Glu Lys Glu Lys Ile Glu Ala Ala Leu Lys Glu Ala Glu Glu Ala 565 570 575 Val Lys Gly Asp Asp Lys Ala Ala Ile Asp Ala Lys Thr Glu Ala Leu 580 585 590 Gly Ala Ala Ser Gln Lys Leu Gly Glu Met Val Tyr Ala Gln Ala Gln 595 600 605 Ala Glu Ala Gln Ala Gly Glu Ser Glu Gln Ala Asn Ala Ser Ala Lys 610 615 620 Lys Asp Asp Asp Val Val Asp Ala Asp Phe Glu Glu Val Lys Asp Asp 625 630 635 640 Lys Lys 14 27 DNA Artificial Sequence Primer Used to clone Candida glabrata Hsp60 gene and to contruct Candida glabrata Hsp60 expression plasmids 14 gatatacata tggccaagga gttgaag 27 15 642 PRT Neisseria meningitidis 15 Met Ala Lys Val Ile Gly Ile Asp Leu Gly Thr Thr Asn Ser Cys Leu 1 5 10 15 Ala Ile Ser Glu Asn Gly Gln Thr Lys Val Ile Glu Asn Ala Glu Gly 20 25 30 Ala Arg Thr Thr Pro Ser Val Ile Ala Tyr Leu Asp Gly Gly Glu Ile 35 40 45 Leu Val Gly Ala Pro Ala Lys Arg Gln Ala Val Thr Asn Ala Lys Asn 50 55 60 Thr Ile Tyr Ala Ala Lys Arg Leu Ile Gly His Lys Phe Glu Asp Lys 65 70 75 80 Glu Val Gln Arg Asp Ile Glu Ser Met Pro Phe Glu Ile Ile Lys Ala 85 90 95 Asn Asn Gly Asp Ala Trp Val Lys Ala Gln Gly Lys Glu Leu Ser Pro 100 105 110 Pro Gln Ile Ser Ala Glu Val Leu Arg Lys Met Lys Glu Ala Ala Glu 115 120 125 Ala Tyr Leu Gly Glu Lys Val Thr Glu Ala Val Ile Thr Val Pro Ala 130 135 140 Tyr Phe Asn Asp Ser Gln Arg Gln Ala Thr Lys Asp Ala Gly Arg Ile 145 150 155 160 Ala Gly Leu Asp Val Lys Arg Ile Ile Asn Glu Pro Thr Ala Ala Ala 165 170 175 Leu Ala Phe Gly Met Asp Lys Gly Asp Asn Lys Asp Arg Lys Val Ala 180 185 190 Val Tyr Asp Leu Gly Gly Gly Thr Phe Asp Ile Ser Ile Ile Glu Ile 195 200 205 Ala Asn Leu Asp Gly Asp Lys Gln Phe Glu Val Leu Ala Thr Asn Gly 210 215 220 Asp Thr Phe Leu Gly Gly Glu Asp Phe Asp Gln Arg Leu Ile Asp His 225 230 235 240 Ile Ile Ala Glu Phe Lys Lys Glu Gln Gly Ile Asp Leu Lys Gln Asp 245 250 255 Val Met Ala Leu Gln Arg Leu Lys Glu Ala Ala Glu Lys Ala Lys Ile 260 265 270 Glu Leu Ser Ser Gly Gln Gln Thr Glu Ile Asn Leu Pro Tyr Ile Thr 275 280 285 Met Asp Ala Thr Gly Pro Lys His Leu Ala Met Lys Ile Thr Arg Ala 290 295 300 Lys Phe Glu Ser Leu Val Glu Asp Leu Ile Thr Arg Ser Ile Glu Pro 305 310 315 320 Cys Lys Ile Ala Leu Lys Asp Ala Gly Leu Ser Thr Gly Asp Ile Asp 325 330 335 Asp Val Ile Leu Val Gly Gly Gln Ser Arg Met Pro Lys Val Gln Glu 340 345 350 Ala Val Lys Ala Phe Phe Gly Lys Glu Pro Arg Lys Asp Val Asn Pro 355 360 365 Asp Glu Ala Val Ala Val Gly Ala Ala Ile Gln Gly Glu Val Leu Ser 370 375 380 Gly Gly Arg Ser Asp Val Leu Leu Leu Asp Val Thr Pro Leu Ser Leu 385 390 395 400 Gly Ile Glu Thr Met Gly Gly Val Met Thr Lys Leu Ile Gln Lys Asn 405 410 415 Thr Thr Ile Pro Thr Lys Ala Ser Gln Val Phe Ser Thr Ala Glu Asp 420 425 430 Asn Gln Ser Ala Val Thr Ile His Val Leu Gln Gly Glu Arg Glu Arg 435 440 445 Ala Ser Ala Asn Lys Ser Leu Gly Gln Phe Asn Leu Gly Asp Ile Ala 450 455 460 Pro Ala Pro Arg Gly Met Pro Gln Ile Glu Val Thr Phe Asp Ile Asp 465 470 475 480 Ala Asn Gly Ile Leu His Val Ser Ala Lys Asp Lys Gly Thr Gly Lys 485 490 495 Ala Ala Asn Ile Thr Ile Gln Gly Ser Ser Gly Leu Ser Glu Glu Glu 500 505 510 Ile Glu Arg Met Val Lys Asp Ala Glu Ala Asn Ala Glu Glu Asp Lys 515 520 525 Lys Leu Thr Glu Leu Val Ala Ser Arg Asn Gln Ala Glu Ala Leu Ile 530 535 540 His Ser Val Lys Lys Ser Leu Ala Asp Tyr Gly Asp Lys Leu Asp Ala 545 550 555 560 Ala Glu Lys Glu Lys Ile Glu Ala Ala Leu Lys Glu Ala Glu Glu Ala 565 570 575 Val Lys Gly Asp Asp Lys Ala Ala Ile Asp Ala Lys Thr Glu Ala Leu 580 585 590 Gly Ala Ala Ser Gln Lys Leu Gly Glu Met Val Tyr Ala Gln Ala Gln 595 600 605 Ala Glu Ala Gln Ala Gly Glu Ser Glu Gln Ala Asn Ala Ser Ala Lys 610

615 620 Lys Asp Asp Asp Val Val Asp Ala Asp Phe Glu Glu Val Lys Asp Asp 625 630 635 640 Lys Lys 16 662 PRT Neisseria meningitidis 16 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Lys Val Ile Gly Ile Asp Leu Gly Thr Thr 20 25 30 Asn Ser Cys Leu Ala Ile Ser Glu Asn Gly Gln Thr Lys Val Ile Glu 35 40 45 Asn Ala Glu Gly Ala Arg Thr Thr Pro Ser Val Ile Ala Tyr Leu Asp 50 55 60 Gly Gly Glu Ile Leu Val Gly Ala Pro Ala Lys Arg Gln Ala Val Thr 65 70 75 80 Asn Ala Lys Asn Thr Ile Tyr Ala Ala Lys Arg Leu Ile Gly His Lys 85 90 95 Phe Glu Asp Lys Glu Val Gln Arg Asp Ile Glu Ser Met Pro Phe Glu 100 105 110 Ile Ile Lys Ala Asn Asn Gly Asp Ala Trp Val Lys Ala Gln Gly Lys 115 120 125 Glu Leu Ser Pro Pro Gln Ile Ser Ala Glu Val Leu Arg Lys Met Lys 130 135 140 Glu Ala Ala Glu Ala Tyr Leu Gly Glu Lys Val Thr Glu Ala Val Ile 145 150 155 160 Thr Val Pro Ala Tyr Phe Asn Asp Ser Gln Arg Gln Ala Thr Lys Asp 165 170 175 Ala Gly Arg Ile Ala Gly Leu Asp Val Lys Arg Ile Ile Asn Glu Pro 180 185 190 Thr Ala Ala Ala Leu Ala Phe Gly Met Asp Lys Gly Asp Asn Lys Asp 195 200 205 Arg Lys Val Ala Val Tyr Asp Leu Gly Gly Gly Thr Phe Asp Ile Ser 210 215 220 Ile Ile Glu Ile Ala Asn Leu Asp Gly Asp Lys Gln Phe Glu Val Leu 225 230 235 240 Ala Thr Asn Gly Asp Thr Phe Leu Gly Gly Glu Asp Phe Asp Gln Arg 245 250 255 Leu Ile Asp His Ile Ile Ala Glu Phe Lys Lys Glu Gln Gly Ile Asp 260 265 270 Leu Lys Gln Asp Val Met Ala Leu Gln Arg Leu Lys Glu Ala Ala Glu 275 280 285 Lys Ala Lys Ile Glu Leu Ser Ser Gly Gln Gln Thr Glu Ile Asn Leu 290 295 300 Pro Tyr Ile Thr Met Asp Ala Thr Gly Pro Lys His Leu Ala Met Lys 305 310 315 320 Ile Thr Arg Ala Lys Phe Glu Ser Leu Val Glu Asp Leu Ile Thr Arg 325 330 335 Ser Ile Glu Pro Cys Lys Ile Ala Leu Lys Asp Ala Gly Leu Ser Thr 340 345 350 Gly Asp Ile Asp Asp Val Ile Leu Val Gly Gly Gln Ser Arg Met Pro 355 360 365 Lys Val Gln Glu Ala Val Lys Ala Phe Phe Gly Lys Glu Pro Arg Lys 370 375 380 Asp Val Asn Pro Asp Glu Ala Val Ala Val Gly Ala Ala Ile Gln Gly 385 390 395 400 Glu Val Leu Ser Gly Gly Arg Ser Asp Val Leu Leu Leu Asp Val Thr 405 410 415 Pro Leu Ser Leu Gly Ile Glu Thr Met Gly Gly Val Met Thr Lys Leu 420 425 430 Ile Gln Lys Asn Thr Thr Ile Pro Thr Lys Ala Ser Gln Val Phe Ser 435 440 445 Thr Ala Glu Asp Asn Gln Ser Ala Val Thr Ile His Val Leu Gln Gly 450 455 460 Glu Arg Glu Arg Ala Ser Ala Asn Lys Ser Leu Gly Gln Phe Asn Leu 465 470 475 480 Gly Asp Ile Ala Pro Ala Pro Arg Gly Met Pro Gln Ile Glu Val Thr 485 490 495 Phe Asp Ile Asp Ala Asn Gly Ile Leu His Val Ser Ala Lys Asp Lys 500 505 510 Gly Thr Gly Lys Ala Ala Asn Ile Thr Ile Gln Gly Ser Ser Gly Leu 515 520 525 Ser Glu Glu Glu Ile Glu Arg Met Val Lys Asp Ala Glu Ala Asn Ala 530 535 540 Glu Glu Asp Lys Lys Leu Thr Glu Leu Val Ala Ser Arg Asn Gln Ala 545 550 555 560 Glu Ala Leu Ile His Ser Val Lys Lys Ser Leu Ala Asp Tyr Gly Asp 565 570 575 Lys Leu Asp Ala Ala Glu Lys Glu Lys Ile Glu Ala Ala Leu Lys Glu 580 585 590 Ala Glu Glu Ala Val Lys Gly Asp Asp Lys Ala Ala Ile Asp Ala Lys 595 600 605 Thr Glu Ala Leu Gly Ala Ala Ser Gln Lys Leu Gly Glu Met Val Tyr 610 615 620 Ala Gln Ala Gln Ala Glu Ala Gln Ala Gly Glu Ser Glu Gln Ala Asn 625 630 635 640 Ala Ser Ala Lys Lys Asp Asp Asp Val Val Asp Ala Asp Phe Glu Glu 645 650 655 Val Lys Asp Asp Lys Lys 660 17 37 PRT aspergillus fumigatus 17 Met Gln Arg Ala Leu Ser Ser Arg Thr Ser Val Leu Ser Ala Ala Ser 1 5 10 15 Lys Arg Ala Ala Phe Thr Lys Pro Ala Gly Leu Asn Leu Gln Gln Gln 20 25 30 Arg Phe Ala His Lys 35 18 48 PRT Aspergillus fumigatus 18 Glu Leu Lys Phe Gly Val Glu Ala Arg Ala Gln Leu Leu Lys Gly Val 1 5 10 15 Asp Thr Leu Ala Lys Ala Val Thr Ser Thr Leu Gly Pro Lys Gly Arg 20 25 30 Asn Val Leu Ile Glu Ser Pro Tyr Gly Ser Pro Lys Ile Thr Lys Asp 35 40 45 19 502 PRT Aspergillus fumigatus 19 Gly Val Ser Val Ala Lys Ala Ile Thr Leu Gln Asp Lys Phe Glu Asn 1 5 10 15 Leu Gly Ala Arg Leu Leu Gln Asp Val Ala Ser Lys Thr Asn Glu Ile 20 25 30 Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala Arg Ala Ile Phe 35 40 45 Ser Glu Thr Val Lys Asn Val Ala Ala Gly Cys Asn Pro Met Asp Leu 50 55 60 Arg Arg Gly Ile Gln Ala Ala Val Asp Ala Val Val Asp Tyr Leu Gln 65 70 75 80 Lys Asn Lys Arg Asp Ile Thr Thr Gly Glu Glu Ile Ala Gln Val Ala 85 90 95 Thr Ile Ser Ala Asn Gly Asp Thr His Ile Gly Lys Leu Ile Ser Thr 100 105 110 Ala Met Glu Arg Val Gly Lys Glu Gly Val Ile Thr Val Lys Glu Gly 115 120 125 Lys Thr Ile Glu Asp Glu Leu Glu Val Thr Glu Gly Met Arg Phe Asp 130 135 140 Arg Gly Tyr Thr Ser Pro Tyr Phe Ile Thr Asp Thr Lys Ser Gln Lys 145 150 155 160 Val Glu Phe Glu Lys Pro Leu Ile Leu Leu Ser Glu Lys Lys Ile Ser 165 170 175 Ala Val Gln Asp Ile Ile Pro Ala Leu Glu Ala Ser Thr Thr Leu Arg 180 185 190 Arg Pro Leu Val Ile Ile Ala Glu Asp Ile Glu Gly Glu Ala Leu Ala 195 200 205 Val Cys Ile Leu Asn Lys Leu Arg Gly Gln Leu Gln Val Ala Ala Val 210 215 220 Lys Ala Pro Gly Phe Gly Asp Asn Arg Lys Ser Ile Leu Gly Asp Leu 225 230 235 240 Ala Val Leu Thr Asn Gly Thr Val Phe Thr Asp Glu Leu Asp Ile Lys 245 250 255 Leu Glu Lys Leu Thr Pro Asp Met Leu Gly Ser Thr Gly Ala Ile Thr 260 265 270 Ile Thr Lys Glu Asp Thr Ile Ile Leu Asn Gly Glu Gly Ser Lys Asp 275 280 285 Ala Ile Ala Gln Arg Cys Glu Gln Ile Arg Gly Val Met Ala Asp Pro 290 295 300 Ser Thr Ser Glu Tyr Glu Lys Glu Lys Leu Gln Glu Arg Leu Ala Lys 305 310 315 320 Leu Ser Gly Gly Val Ala Val Ile Lys Val Gly Gly Ala Ser Glu Val 325 330 335 Glu Val Gly Glu Lys Lys Asp Arg Val Val Asp Ala Leu Asn Ala Thr 340 345 350 Arg Ala Ala Val Glu Glu Gly Ile Leu Pro Gly Gly Gly Thr Ala Leu 355 360 365 Leu Lys Ala Ala Ala Asn Gly Leu Asp Asn Val Lys Pro Glu Asn Phe 370 375 380 Asp Gln Gln Leu Gly Val Ser Ile Ile Lys Asn Ala Ile Thr Arg Pro 385 390 395 400 Ala Arg Thr Ile Val Glu Asn Ala Gly Leu Glu Gly Ser Val Ile Val 405 410 415 Gly Lys Leu Thr Asp Glu Phe Ala Lys Asp Phe Asn Arg Gly Phe Asp 420 425 430 Ser Ser Lys Gly Glu Tyr Val Asp Met Ile Ser Ser Gly Ile Leu Asp 435 440 445 Pro Leu Lys Val Val Arg Thr Ala Leu Leu Asp Ala Ser Gly Val Ala 450 455 460 Ser Leu Leu Gly Thr Thr Glu Val Ala Ile Val Glu Ala Pro Glu Glu 465 470 475 480 Lys Gly Pro Ala Ala Pro Gly Met Gly Gly Met Gly Gly Met Gly Gly 485 490 495 Met Gly Gly Gly Met Phe 500 20 550 PRT Aspergillus fumigatus 20 Met Lys Glu Leu Lys Phe Gly Val Glu Ala Arg Ala Gln Leu Leu Lys 1 5 10 15 Gly Val Asp Thr Leu Ala Lys Ala Val Thr Ser Thr Leu Gly Pro Lys 20 25 30 Gly Arg Asn Val Leu Ile Glu Ser Pro Tyr Gly Ser Pro Lys Ile Thr 35 40 45 Lys Asp Gly Val Ser Val Ala Lys Ala Ile Thr Leu Gln Asp Lys Phe 50 55 60 Glu Asn Leu Gly Ala Arg Leu Leu Gln Asp Val Ala Ser Lys Thr Asn 65 70 75 80 Glu Ile Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala Arg Ala 85 90 95 Ile Phe Ser Glu Thr Val Lys Asn Val Ala Ala Gly Cys Asn Pro Met 100 105 110 Asp Leu Arg Arg Gly Ile Gln Ala Ala Val Asp Ala Val Val Asp Tyr 115 120 125 Leu Gln Lys Asn Lys Arg Asp Ile Thr Thr Gly Glu Glu Ile Ala Gln 130 135 140 Val Ala Thr Ile Ser Ala Asn Gly Asp Thr His Ile Gly Lys Leu Ile 145 150 155 160 Ser Thr Ala Met Glu Arg Val Gly Lys Glu Gly Val Ile Thr Val Lys 165 170 175 Glu Gly Lys Thr Ile Glu Asp Glu Leu Glu Val Thr Glu Gly Met Arg 180 185 190 Phe Asp Arg Gly Tyr Thr Ser Pro Tyr Phe Ile Thr Asp Thr Lys Ser 195 200 205 Gln Lys Val Glu Phe Glu Lys Pro Leu Ile Leu Leu Ser Glu Lys Lys 210 215 220 Ile Ser Ala Val Gln Asp Ile Ile Pro Ala Leu Glu Ala Ser Thr Thr 225 230 235 240 Leu Arg Arg Pro Leu Val Ile Ile Ala Glu Asp Ile Glu Gly Glu Ala 245 250 255 Leu Ala Val Cys Ile Leu Asn Lys Leu Arg Gly Gln Leu Gln Val Ala 260 265 270 Ala Val Lys Ala Pro Gly Phe Gly Asp Asn Arg Lys Ser Ile Leu Gly 275 280 285 Asp Leu Ala Val Leu Thr Asn Gly Thr Val Phe Thr Asp Glu Leu Asp 290 295 300 Ile Lys Leu Glu Lys Leu Thr Pro Asp Met Leu Gly Ser Thr Gly Ala 305 310 315 320 Ile Thr Ile Thr Lys Glu Asp Thr Ile Ile Leu Asn Gly Glu Gly Ser 325 330 335 Lys Asp Ala Ile Ala Gln Arg Cys Glu Gln Ile Arg Gly Val Met Ala 340 345 350 Asp Pro Ser Thr Ser Glu Tyr Glu Lys Glu Lys Leu Gln Glu Arg Leu 355 360 365 Ala Lys Leu Ser Gly Gly Val Ala Val Ile Lys Val Gly Gly Ala Ser 370 375 380 Glu Val Glu Val Gly Glu Lys Lys Asp Arg Val Val Asp Ala Leu Asn 385 390 395 400 Ala Thr Arg Ala Ala Val Glu Glu Gly Ile Leu Pro Gly Gly Gly Thr 405 410 415 Ala Leu Leu Lys Ala Ala Ala Asn Gly Leu Asp Asn Val Lys Pro Glu 420 425 430 Asn Phe Asp Gln Gln Leu Gly Val Ser Ile Ile Lys Asn Ala Ile Thr 435 440 445 Arg Pro Ala Arg Thr Ile Val Glu Asn Ala Gly Leu Glu Gly Ser Val 450 455 460 Ile Val Gly Lys Leu Thr Asp Glu Phe Ala Lys Asp Phe Asn Arg Gly 465 470 475 480 Phe Asp Ser Ser Lys Gly Glu Tyr Val Asp Met Ile Ser Ser Gly Ile 485 490 495 Leu Asp Pro Leu Lys Val Val Arg Thr Ala Leu Leu Asp Ala Ser Gly 500 505 510 Val Ala Ser Leu Leu Gly Thr Thr Glu Val Ala Ile Val Glu Ala Pro 515 520 525 Glu Glu Lys Gly Pro Ala Ala Pro Gly Met Gly Gly Met Gly Gly Met 530 535 540 Gly Gly Met Gly Gly Met 545 550 21 570 PRT Aspergillus fumigatus 21 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Lys Glu Leu Lys Phe Gly Val Glu Ala Arg Ala 20 25 30 Gln Leu Leu Lys Gly Val Asp Thr Leu Ala Lys Ala Val Thr Ser Thr 35 40 45 Leu Gly Pro Lys Gly Arg Asn Val Leu Ile Glu Ser Pro Tyr Gly Ser 50 55 60 Pro Lys Ile Thr Lys Asp Gly Val Ser Val Ala Lys Ala Ile Thr Leu 65 70 75 80 Gln Asp Lys Phe Glu Asn Leu Gly Ala Arg Leu Leu Gln Asp Val Ala 85 90 95 Ser Lys Thr Asn Glu Ile Ala Gly Asp Gly Thr Thr Thr Ala Thr Val 100 105 110 Leu Ala Arg Ala Ile Phe Ser Glu Thr Val Lys Asn Val Ala Ala Gly 115 120 125 Cys Asn Pro Met Asp Leu Arg Arg Gly Ile Gln Ala Ala Val Asp Ala 130 135 140 Val Val Asp Tyr Leu Gln Lys Asn Lys Arg Asp Ile Thr Thr Gly Glu 145 150 155 160 Glu Ile Ala Gln Val Ala Thr Ile Ser Ala Asn Gly Asp Thr His Ile 165 170 175 Gly Lys Leu Ile Ser Thr Ala Met Glu Arg Val Gly Lys Glu Gly Val 180 185 190 Ile Thr Val Lys Glu Gly Lys Thr Ile Glu Asp Glu Leu Glu Val Thr 195 200 205 Glu Gly Met Arg Phe Asp Arg Gly Tyr Thr Ser Pro Tyr Phe Ile Thr 210 215 220 Asp Thr Lys Ser Gln Lys Val Glu Phe Glu Lys Pro Leu Ile Leu Leu 225 230 235 240 Ser Glu Lys Lys Ile Ser Ala Val Gln Asp Ile Ile Pro Ala Leu Glu 245 250 255 Ala Ser Thr Thr Leu Arg Arg Pro Leu Val Ile Ile Ala Glu Asp Ile 260 265 270 Glu Gly Glu Ala Leu Ala Val Cys Ile Leu Asn Lys Leu Arg Gly Gln 275 280 285 Leu Gln Val Ala Ala Val Lys Ala Pro Gly Phe Gly Asp Asn Arg Lys 290 295 300 Ser Ile Leu Gly Asp Leu Ala Val Leu Thr Asn Gly Thr Val Phe Thr 305 310 315 320 Asp Glu Leu Asp Ile Lys Leu Glu Lys Leu Thr Pro Asp Met Leu Gly 325 330 335 Ser Thr Gly Ala Ile Thr Ile Thr Lys Glu Asp Thr Ile Ile Leu Asn 340 345 350 Gly Glu Gly Ser Lys Asp Ala Ile Ala Gln Arg Cys Glu Gln Ile Arg 355 360 365 Gly Val Met Ala Asp Pro Ser Thr Ser Glu Tyr Glu Lys Glu Lys Leu 370 375 380 Gln Glu Arg Leu Ala Lys Leu Ser Gly Gly Val Ala Val Ile Lys Val 385 390 395 400 Gly Gly Ala Ser Glu Val Glu Val Gly Glu Lys Lys Asp Arg Val Val 405 410 415 Asp Ala Leu Asn Ala Thr Arg Ala Ala Val Glu Glu Gly Ile Leu Pro 420 425 430 Gly Gly Gly Thr Ala Leu Leu Lys Ala Ala Ala Asn Gly Leu Asp Asn 435 440 445 Val Lys Pro Glu Asn Phe Asp Gln Gln Leu Gly Val Ser Ile Ile Lys 450 455 460 Asn Ala Ile Thr Arg Pro Ala Arg Thr Ile Val Glu Asn Ala Gly Leu 465 470 475 480 Glu Gly Ser Val Ile Val Gly Lys Leu Thr Asp Glu Phe Ala Lys Asp 485 490 495 Phe Asn Arg Gly Phe Asp Ser Ser Lys Gly Glu Tyr Val Asp Met Ile 500 505 510 Ser Ser Gly Ile Leu Asp Pro Leu Lys Val Val Arg Thr Ala Leu Leu 515 520 525 Asp Ala Ser Gly Val Ala Ser Leu Leu Gly Thr Thr Glu Val Ala Ile 530 535 540 Val Glu Ala Pro Glu Glu Lys Gly Pro Ala Ala Pro Gly Met Gly Gly 545 550 555 560 Met Gly Gly Met Gly Gly Met Gly Gly Met 565 570 22 568 PRT Candida glabrata 22 Met Leu Arg Ala Val Ala Arg Ser Gln Val Arg Ser Leu Arg Asn Ala 1 5 10 15 Arg Leu Tyr Ser Ser Phe Lys Glu Leu Lys Phe Gly Val Glu Gly Arg

20 25 30 Ala Ala Leu Leu Arg Gly Val Glu Thr Leu Ala Asp Ala Val Ser Ala 35 40 45 Thr Leu Gly Pro Lys Gly Arg Asn Val Leu Ile Glu Gln Pro Phe Gly 50 55 60 Ala Pro Lys Ile Thr Lys Asp Gly Val Thr Val Ala Arg Ser Ile Thr 65 70 75 80 Leu Glu Asp Lys Phe Glu Asn Met Gly Ala Lys Leu Leu Gln Glu Val 85 90 95 Ala Ser Lys Thr Asn Glu Ala Ala Gly Asp Gly Thr Thr Ser Ala Thr 100 105 110 Val Leu Gly Arg Ala Ile Phe Thr Glu Ser Val Lys Asn Val Ala Ala 115 120 125 Gly Cys Asn Pro Met Asp Leu Arg Arg Gly Ser Gln Ala Ala Val Glu 130 135 140 Lys Val Ile Gln Phe Leu Thr Glu Asn Lys Lys Glu Ile Thr Thr Ser 145 150 155 160 Glu Glu Ile Ala Gln Val Ala Thr Ile Ser Ala Asn Gly Asp Ala His 165 170 175 Val Gly Lys Leu Leu Ala Ser Ala Met Glu Lys Val Gly Lys Glu Gly 180 185 190 Val Ile Thr Ile Arg Glu Gly Arg Thr Leu Glu Asp Glu Leu Glu Val 195 200 205 Thr Glu Gly Met Arg Phe Asp Arg Gly Phe Ile Ser Pro Tyr Phe Ile 210 215 220 Thr Asp Ala Lys Ser Gly Lys Val Glu Phe Glu Lys Pro Leu Leu Leu 225 230 235 240 Leu Ser Glu Lys Lys Ile Ser Ser Ile Gln Asp Ile Leu Pro Ala Leu 245 250 255 Glu Leu Ser Asn Gln Ser Arg Arg Pro Leu Leu Ile Ile Ala Glu Asp 260 265 270 Val Asp Gly Glu Ala Leu Ala Ala Cys Ile Leu Asn Lys Leu Arg Gly 275 280 285 Gln Val Lys Val Cys Ala Val Lys Ala Pro Gly Phe Gly Asp Asn Arg 290 295 300 Lys Asn Ile Leu Gly Asp Val Ala Ile Leu Thr Gly Ser Thr Val Phe 305 310 315 320 Thr Glu Glu Leu Asp Leu Lys Pro Glu Gln Ala Thr Met Glu His Leu 325 330 335 Gly Ser Cys Asp Ser Ile Thr Ile Thr Lys Glu Asp Thr Val Ile Leu 340 345 350 Asn Gly Asn Gly Ser Lys Asp Ser Ile Gln Glu Arg Ile Glu Gln Ile 355 360 365 Lys Asn Ser Ile Asp Val Thr Thr Thr Asn Ser Tyr Glu Lys Glu Lys 370 375 380 Leu Gln Glu Arg Leu Ala Lys Leu Ser Gly Gly Val Ala Val Ile Arg 385 390 395 400 Val Gly Gly Ala Ser Glu Val Glu Val Gly Glu Lys Lys Asp Arg Tyr 405 410 415 Asp Asp Ala Leu Asn Ala Thr Arg Ala Ala Val Glu Glu Gly Ile Leu 420 425 430 Pro Gly Gly Gly Thr Ala Leu Val Lys Ala Ser Arg Val Leu Asp Glu 435 440 445 Val Lys Thr Glu Asn Phe Asp Gln Lys Leu Gly Val Asp Ile Ile Arg 450 455 460 Lys Ala Ile Thr Arg Pro Ala Lys Gln Ile Ile Glu Asn Ala Gly Glu 465 470 475 480 Glu Gly Ser Val Ile Val Gly Lys Leu Val Asp Glu Phe Gly Glu Asp 485 490 495 Phe Ala Lys Gly Tyr Asp Ser Ala Lys Gly Glu Phe Thr Asp Met Leu 500 505 510 Ala Ala Gly Ile Ile Asp Pro Phe Lys Val Val Arg Ser Gly Leu Val 515 520 525 Asp Ala Ser Gly Val Ala Ser Leu Leu Ala Thr Thr Glu Val Ala Ile 530 535 540 Val Asp Ala Pro Glu Pro Ala Pro Ala Ala Gly Ala Pro Gly Gly Gly 545 550 555 560 Met Pro Gly Met Pro Gly Met Met 565 23 548 PRT Candida glabrata 23 Met Ala Lys Glu Leu Lys Phe Gly Val Glu Gly Arg Ala Ala Leu Leu 1 5 10 15 Arg Gly Val Glu Thr Leu Ala Asp Ala Val Ser Ala Thr Leu Gly Pro 20 25 30 Lys Gly Arg Asn Val Leu Ile Glu Gln Pro Phe Gly Ala Pro Lys Ile 35 40 45 Thr Lys Asp Gly Val Thr Val Ala Arg Ser Ile Thr Leu Glu Asp Lys 50 55 60 Phe Glu Asn Met Gly Ala Lys Leu Leu Gln Glu Val Ala Ser Lys Thr 65 70 75 80 Asn Glu Ala Ala Gly Asp Gly Thr Thr Ser Ala Thr Val Leu Gly Arg 85 90 95 Ala Ile Phe Thr Glu Ser Val Lys Asn Val Ala Ala Gly Cys Asn Pro 100 105 110 Met Asp Leu Arg Arg Gly Ser Gln Ala Ala Val Glu Lys Val Ile Gln 115 120 125 Phe Leu Thr Glu Asn Lys Lys Glu Ile Thr Thr Ser Glu Glu Ile Ala 130 135 140 Gln Val Ala Thr Ile Ser Ala Asn Gly Asp Ala His Val Gly Lys Leu 145 150 155 160 Leu Ala Ser Ala Met Glu Lys Val Gly Lys Glu Gly Val Ile Thr Ile 165 170 175 Arg Glu Gly Arg Thr Leu Glu Asp Glu Leu Glu Val Thr Glu Gly Met 180 185 190 Arg Phe Asp Arg Gly Phe Ile Ser Pro Tyr Phe Ile Thr Asp Ala Lys 195 200 205 Ser Gly Lys Val Glu Phe Glu Lys Pro Leu Leu Leu Leu Ser Glu Lys 210 215 220 Lys Ile Ser Ser Ile Gln Asp Ile Leu Pro Ala Leu Glu Leu Ser Asn 225 230 235 240 Gln Ser Arg Arg Pro Leu Leu Ile Ile Ala Glu Asp Val Asp Gly Glu 245 250 255 Ala Leu Ala Ala Cys Ile Leu Asn Lys Leu Arg Gly Gln Val Lys Val 260 265 270 Cys Ala Val Lys Ala Pro Gly Phe Gly Asp Asn Arg Lys Asn Ile Leu 275 280 285 Gly Asp Val Ala Ile Leu Thr Gly Ser Thr Val Phe Thr Glu Glu Leu 290 295 300 Asp Leu Lys Pro Glu Gln Ala Thr Met Glu His Leu Gly Ser Cys Asp 305 310 315 320 Ser Ile Thr Ile Thr Lys Glu Asp Thr Val Ile Leu Asn Gly Asn Gly 325 330 335 Ser Lys Asp Ser Ile Gln Glu Arg Ile Glu Gln Ile Lys Asn Ser Ile 340 345 350 Asp Val Thr Thr Thr Asn Ser Tyr Glu Lys Glu Lys Leu Gln Glu Arg 355 360 365 Leu Ala Lys Leu Ser Gly Gly Val Ala Val Ile Arg Val Gly Gly Ala 370 375 380 Ser Glu Val Glu Val Gly Glu Lys Lys Asp Arg Tyr Asp Asp Ala Leu 385 390 395 400 Asn Ala Thr Arg Ala Ala Val Glu Glu Gly Ile Leu Pro Gly Gly Gly 405 410 415 Thr Ala Leu Val Lys Ala Ser Arg Val Leu Asp Glu Val Lys Thr Glu 420 425 430 Asn Phe Asp Gln Lys Leu Gly Val Asp Ile Ile Arg Lys Ala Ile Thr 435 440 445 Arg Pro Ala Lys Gln Ile Ile Glu Asn Ala Gly Glu Glu Gly Ser Val 450 455 460 Ile Val Gly Lys Leu Val Asp Glu Phe Gly Glu Asp Phe Ala Lys Gly 465 470 475 480 Tyr Asp Ser Ala Lys Gly Glu Phe Thr Asp Met Leu Ala Ala Gly Ile 485 490 495 Ile Asp Pro Phe Lys Val Val Arg Ser Gly Leu Val Asp Ala Ser Gly 500 505 510 Val Ala Ser Leu Leu Ala Thr Thr Glu Val Ala Ile Val Asp Ala Pro 515 520 525 Glu Pro Ala Pro Ala Ala Gly Ala Pro Gly Gly Gly Met Pro Gly Met 530 535 540 Pro Gly Met Met 545 24 568 PRT Candida glabrata 24 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Lys Glu Leu Lys Phe Gly Val Glu Gly Arg 20 25 30 Ala Ala Leu Leu Arg Gly Val Glu Thr Leu Ala Asp Ala Val Ser Ala 35 40 45 Thr Leu Gly Pro Lys Gly Arg Asn Val Leu Ile Glu Gln Pro Phe Gly 50 55 60 Ala Pro Lys Ile Thr Lys Asp Gly Val Thr Val Ala Arg Ser Ile Thr 65 70 75 80 Leu Glu Asp Lys Phe Glu Asn Met Gly Ala Lys Leu Leu Gln Glu Val 85 90 95 Ala Ser Lys Thr Asn Glu Ala Ala Gly Asp Gly Thr Thr Ser Ala Thr 100 105 110 Val Leu Gly Arg Ala Ile Phe Thr Glu Ser Val Lys Asn Val Ala Ala 115 120 125 Gly Cys Asn Pro Met Asp Leu Arg Arg Gly Ser Gln Ala Ala Val Glu 130 135 140 Lys Val Ile Gln Phe Leu Thr Glu Asn Lys Lys Glu Ile Thr Thr Ser 145 150 155 160 Glu Glu Ile Ala Gln Val Ala Thr Ile Ser Ala Asn Gly Asp Ala His 165 170 175 Val Gly Lys Leu Leu Ala Ser Ala Met Glu Lys Val Gly Lys Glu Gly 180 185 190 Val Ile Thr Ile Arg Glu Gly Arg Thr Leu Glu Asp Glu Leu Glu Val 195 200 205 Thr Glu Gly Met Arg Phe Asp Arg Gly Phe Ile Ser Pro Tyr Phe Ile 210 215 220 Thr Asp Ala Lys Ser Gly Lys Val Glu Phe Glu Lys Pro Leu Leu Leu 225 230 235 240 Leu Ser Glu Lys Lys Ile Ser Ser Ile Gln Asp Ile Leu Pro Ala Leu 245 250 255 Glu Leu Ser Asn Gln Ser Arg Arg Pro Leu Leu Ile Ile Ala Glu Asp 260 265 270 Val Asp Gly Glu Ala Leu Ala Ala Cys Ile Leu Asn Lys Leu Arg Gly 275 280 285 Gln Val Lys Val Cys Ala Val Lys Ala Pro Gly Phe Gly Asp Asn Arg 290 295 300 Lys Asn Ile Leu Gly Asp Val Ala Ile Leu Thr Gly Ser Thr Val Phe 305 310 315 320 Thr Glu Glu Leu Asp Leu Lys Pro Glu Gln Ala Thr Met Glu His Leu 325 330 335 Gly Ser Cys Asp Ser Ile Thr Ile Thr Lys Glu Asp Thr Val Ile Leu 340 345 350 Asn Gly Asn Gly Ser Lys Asp Ser Ile Gln Glu Arg Ile Glu Gln Ile 355 360 365 Lys Asn Ser Ile Asp Val Thr Thr Thr Asn Ser Tyr Glu Lys Glu Lys 370 375 380 Leu Gln Glu Arg Leu Ala Lys Leu Ser Gly Gly Val Ala Val Ile Arg 385 390 395 400 Val Gly Gly Ala Ser Glu Val Glu Val Gly Glu Lys Lys Asp Arg Tyr 405 410 415 Asp Asp Ala Leu Asn Ala Thr Arg Ala Ala Val Glu Glu Gly Ile Leu 420 425 430 Pro Gly Gly Gly Thr Ala Leu Val Lys Ala Ser Arg Val Leu Asp Glu 435 440 445 Val Lys Thr Glu Asn Phe Asp Gln Lys Leu Gly Val Asp Ile Ile Arg 450 455 460 Lys Ala Ile Thr Arg Pro Ala Lys Gln Ile Ile Glu Asn Ala Gly Glu 465 470 475 480 Glu Gly Ser Val Ile Val Gly Lys Leu Val Asp Glu Phe Gly Glu Asp 485 490 495 Phe Ala Lys Gly Tyr Asp Ser Ala Lys Gly Glu Phe Thr Asp Met Leu 500 505 510 Ala Ala Gly Ile Ile Asp Pro Phe Lys Val Val Arg Ser Gly Leu Val 515 520 525 Asp Ala Ser Gly Val Ala Ser Leu Leu Ala Thr Thr Glu Val Ala Ile 530 535 540 Val Asp Ala Pro Glu Pro Ala Pro Ala Ala Gly Ala Pro Gly Gly Gly 545 550 555 560 Met Pro Gly Met Pro Gly Met Met 565 25 19 DNA Artificial Sequence Primer Used to clone Neisseria meningitidis Hsp70 gene and to contruct Neisseria meningitidis Hsp70 expression vectors 25 ctgccgtaca tcaccatgg 19 26 19 DNA Artificial Sequence Primer Used to clone Neisseria meningitidis Hsp70 gene and to contruct Neisseria meningitidis Hsp70 expression vectors 26 ggcttcttgt actttcggc 19 27 18 DNA Artificial Sequence Primer Used to clone Neisseria meningitidis Hsp70 gene and to contruct Neisseria meningitidis Hsp70 expression vectors 27 tgaccttgtt gaacgtac 18 28 17 DNA Artificial Sequence Primer Used to clone Neisseria meningitidis Hsp70 gene and to contruct Neisseria meningitidis Hsp70 expression vectors 28 acttcatcag ggtttac 17 29 6 PRT Neisseria meningitidis 29 Pro Ala Tyr Phe Asn Asp 1 5 30 18 DNA Artificial Sequence Primer Used to clone Neisseria meningitidis Hsp70 gene and to contruct Neisseria meningitidis Hsp70 expression vectors misc_feature (1)...(18) n = A,T,C or G 30 ccngcntayt tyaaygay 18 31 7 PRT Neisseria meningitidis 31 Pro Gln Ile Glu Val Thr Phe 1 5 32 21 DNA Artificial Sequence Primer Used to clone Neisseria meningitidis Hsp70 gene and to contruct Neisseria meningitidis Hsp70 expression vectors misc_feature (1)...(21) n = A,T,C or G 32 raangtnacy tcdatytgng g 21 33 16 DNA Artificial Sequence Primer Used to clone Neisseria meningitidis Hsp70 gene and to contruct Neisseria meningitidis Hsp70 expression vectors 33 gtaaaacgac ggccag 16 34 17 DNA Artificial Sequence Primer Used to clone Neisseria meningitidis Hsp70 gene and to contruct Neisseria meningitidis Hsp70 expression vectors 34 caggaaacag ctatgac 17 35 27 DNA Artificial Sequence Primer Used to clone Neisseria meningitidis Hsp70 gene and to contruct Neisseria meningitidis Hsp70 expression vectors 35 ggtcggctcg ttgatgatgc gtttcac 27 36 27 DNA Artificial Sequence Primer Used to clone Neisseria meningitidis Hsp70 gene and to contruct Neisseria meningitidis Hsp70 expression vectors 36 gcttctgcca acaaatcttt gggtcag 27 37 24 DNA Artificial Sequence Primer Used to clone Neisseria meningitidis Hsp70 gene and to contruct Neisseria meningitidis Hsp70 expression vectors 37 gccgctttgg cattcgttat ggac 24 38 24 DNA Artificial Sequence Primer Used to clone Neisseria meningitidis Hsp70 gene and to contruct Neisseria meningitidis Hsp70 expression vectors 38 gcgttcgcgt tcgccttgca gtac 24 39 18 DNA Artificial Sequence Primer Used to clone Neisseria meningitidis Hsp70 gene and to contruct Neisseria meningitidis Hsp70 expression vectors 39 ttccgaaaac ggtcaaac 18 40 18 DNA Artificial Sequence Primer Used to clone Neisseria meningitidis Hsp70 gene and to contruct Neisseria meningitidis Hsp70 expression vectors 40 atggccaaac aagagttg 18 41 26 DNA Artificial Sequence Primer Used to clone Neisseria meningitidis Hsp70 gene and to contruct Neisseria meningitidis Hsp70 expression vectors 41 tacatatggc aaaagtaatc ggtatc 26 42 22 DNA Artificial Sequence Primer Used to clone Neisseria meningitidis Hsp70 gene and to contruct Neisseria meningitidis Hsp70 expression vectors 42 tttatttttt gtcgtctttt ac 22 43 19 DNA Artificial Sequence Primer Used to clone Neisseria meningitidis Hsp70 gene and to contruct Neisseria meningitidis Hsp70 expression vectors 43 gtccaaataa gcgataacg 19 44 18 DNA Artificial Sequence Primer Used to clone Neisseria meningitidis Hsp70 gene and to contruct Neisseria meningitidis Hsp70 expression vectors 44 gccgccaaac gtttgatc 18 45 18 DNA Artificial Sequence Primer Used to clone Neisseria meningitidis Hsp70 gene and to contruct Neisseria meningitidis Hsp70 expression vectors 45 accatgggcg gcgtgatg 18 46 18 DNA Artificial Sequence Primer Used to clone Neisseria meningitidis Hsp70 gene and to contruct Neisseria meningitidis Hsp70 expression vectors 46 gaagccaatg ccgaggaa 18 47 18 DNA Artificial Sequence Primer Used to clone Neisseria meningitidis Hsp70 gene and to contruct Neisseria meningitidis Hsp70 expression vectors 47 tgcgtcgccg ttgttggc 18 48 18 DNA Artificial Sequence Primer Used to clone Neisseria meningitidis Hsp70 gene and to contruct Neisseria meningitidis Hsp70 expression vectors 48 ggtatcgccg ttggttgc 18 49 18 DNA Artificial Sequence Primer Used to clone Neisseria meningitidis Hsp70 gene and to contruct Neisseria meningitidis Hsp70 expression vectors 49 gagtttgtcg ccgtagtc 18 50 27 DNA Artificial Sequence Primer Used to clone

Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids misc_feature (1)...(27) n = A,T,C or G 50 ccatatgaar ganytnaart tyggngt 27 51 20 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids modified_base (3)...(3) inosine modified_base (6)...(6) inosine modified_base (9)...(9) inosine modified_base (12)...(12) inosine modified_base (18)...(18) inosine 51 aanganttna antttggngt 20 52 22 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids misc_feature (1)...(22) n = A,T,C or G 52 cttacatcat nccnggcatn cc 22 53 19 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids modified_base (8)...(8) inosine modified_base (11)...(11) inosine modified_base (17)...(17) inosine 53 acatcatncc nggcatncc 19 54 25 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids misc_feature (1)...(25) n = A,T,C or G 54 cttacatncc ncccatnccn cccat 25 55 18 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids modified_base (4)...(4) inosine modified_base (7)...(7) I modified_base (13)...(13) inosine modified_base (16)...(16) inosine 55 catnccnccc atnccncc 18 56 20 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids modified_base (3)...(3) inosine modified_base (6)...(6) inosine modified_base (12)...(12) inosine modified_base (15)...(15) inosine modified_base (18)...(18) inosine 56 gcnggngayg gnacnacnac 20 57 23 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids 57 ggwccmaagg ghmgwaatgt ytt 23 58 23 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids misc_feature (1)...(23) n = A,T,C or G 58 ccnaaratya ctaaggaygg tgt 23 59 20 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids misc_feature (1)...(20) n = A,T,C or G 59 aarganttna aattyggygt 20 60 20 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids misc_feature (1)...(20) n = A,T,C or G 60 tccatnggrt trcanccngc 20 61 20 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids misc_feature (1)...(20) n = A,T,C or G 61 atnacnccyt cyttnccnac 20 62 18 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids misc_feature (1)...(18) n = A,T,C or G 62 catnccytcn gtnacytc 18 63 23 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids 63 acygartgtg cyattgtyga tgc 23 64 23 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids 64 acygargttg cyattgtyga tgc 23 65 23 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids 65 ttagttgatg cttctggtgt ygc 23 66 23 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids 66 ttagttgatg ctagyggtgt ygc 23 67 20 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids misc_feature (1)...(20) n = A,T,C or G 67 garaargara arytncarga 20 68 20 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids misc_feature (1)...(20) n = A,T,C or G 68 gcngcngtng argarggnat 20 69 27 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids 69 ttacatgccg cccatgccgc ccatacc 27 70 24 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids 70 ttacatcata cctggcatac ctgg 24 71 18 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids 71 ccggtggtga tgtcacgc 18 72 18 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids 72 ttgatgacgg caacaccg 18 73 18 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids 73 aactcgtcgg tcagcttg 18 74 18 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids 74 agaacctcgg tgctcgcc 18 75 18 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids 75 cgccatggag cgtgttgg 18 76 18 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids 76 tgctgttgag gagggtat 18 77 18 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids 77 atgatgtcct gaacggca 18 78 18 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids 78 ctgggcgatc ttgccgtc 18 79 21 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids 79 ggtcgtaacg tccttatcga g 21 80 20 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids 80 agagtcgaag tcacggcctt 20 81 22 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids 81 cctcaacaat agcgacctca gt 22 82 18 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids 82 ccccgctgct cctggcat 18 83 19 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids 83 tcgggcagta gtgttcatc 19 84 33 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids 84 tttctcttct atccttggtg atcttagggg agc 33 85 31 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids 85 tttctcttca gatggtgtct ctgttgccaa g 31 86 27 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids 86 ttggattcta catcatacct ggcatac 27 87 19 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids 87 taatacgact cactatagg 19 88 19 DNA Artificial Sequence Primer Used to clone Aspergillus fumigatus Hsp60 gene and to contruct Aspergillus fumigatus Hsp60 expression plasmids 88 gctagttatt gctcagcgg 19 89 18 DNA Artificial Sequence Primer Used to clone Candida glabrata Hsp60 gene and to contruct Candida glabrata Hsp60 expression plasmids 89 cctatggatt tgagaagg 18 90 18 DNA Artificial Sequence Primer Used to clone Candida glabrata Hsp60 gene and to contruct Candida glabrata Hsp60 expression plasmids 90 ctgataatgt caactccc 18 91 19 DNA Artificial Sequence Primer Used to clone Candida glabrata Hsp60 gene and to contruct Candida glabrata Hsp60 expression plasmids 91 gatctcttcc atccaagac 19 92 18 DNA Artificial Sequence Primer Used to clone Candida glabrata Hsp60 gene and to contruct Candida glabrata Hsp60 expression plasmids 92 gtccttggag ccgttacc 18 93 18 DNA Artificial Sequence Primer Used to clone Candida glabrata Hsp60 gene and to contruct Candida glabrata Hsp60 expression plasmids 93 ggtaacggct ccaaggac 18 94 19 DNA Artificial Sequence Primer Used to clone Candida glabrata Hsp60 gene and to contruct Candida glabrata Hsp60 expression plasmids 94 gtcttggatg gaagagatc 19 95 18 DNA Artificial Sequence Primer Used to clone Candida glabrata Hsp60 gene and to contruct Candida glabrata Hsp60 expression plasmids 95 ccttctcaaa tccatagg 18 96 18 DNA Artificial Sequence Primer Used to clone Candida glabrata Hsp60 gene and to contruct Candida glabrata Hsp60 expression plasmids 96 gggagttgac attatcag 18 97 24 DNA Artificial Sequence Primer Used to clone Candida glabrata Hsp60 gene and to contruct Candida glabrata Hsp60 expression plasmids 97 gttgcttcct tgttggctac tacc 24 98 24 DNA Artificial Sequence Primer Used to clone Candida glabrata Hsp60 gene and to contruct Candida glabrata Hsp60 expression plasmids 98 ccccagcgtg gcagagacag cgtc 24 99 24 DNA Artificial Sequence Primer Used to clone Candida glabrata Hsp60 gene and to contruct Candida glabrata Hsp60 expression plasmids 99 gagaacatgg gtgctaagct tctg 24 100 22 DNA Artificial Sequence Primer Used to clone Candida glabrata Hsp60 gene and to contruct Candida glabrata Hsp60 expression plasmids 100 cagctctgcc ttcgacaccg aa 22

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