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  Biologically active synthetic thyrotropin and cloned gene for producing sameUSPTO Application #: 20070190577Title: Biologically active synthetic thyrotropin and cloned gene for producing same Abstract: Substantially pure recombinant TSH has been prepared from a clone comprising complete nucleotide sequence for the expression of the TSH. Diagnostic and therapeutic applications of the synthetic TSH are described. (end of abstract) Agent: Knobbe, Martens, Olson & Bear, LLP - Irvine, CA, US Inventors: Fredric E. Wondisford, Bruce D. Weintraub USPTO Applicaton #: 20070190577 - Class: 435007100 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Antigen-antibody Binding, Specific Binding Protein Assay Or Specific Ligand-receptor Binding Assay The Patent Description & Claims data below is from USPTO Patent Application 20070190577. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATIONS [0001] This application is a continuation of application Ser. No. 11/543,498 filed Oct. 5, 2006, which is a is a continuation of application Ser. No. 11/376,778 filed Mar. 15, 2006, now abandoned, which is a continuation of application Ser. No. 11/148,604 filed Jun. 9, 2005, now abandoned, which is a continuation of application Ser. No. 10/737,469 filed Dec. 16, 2003, now abandoned, which is a continuation of application Ser. No. 09/892,266 filed Jun. 27, 2001, now abandoned, which is a continuation of application Ser. No. 09/569,141 filed May 11, 2000, now U.S. Pat. No. 6,284,491, which is a continuation of application Ser. No. 08/310,923 filed Sep. 22, 1994, now U.S. Pat. No. 6,117,991, which is a continuation of application Ser. No. 08/110,639 filed Aug. 23, 1993, now abandoned, which is a continuation of application Ser. No. 07/882,231 filed May 8, 1992, now abandoned, which is a continuation of application Ser. No. 07/295,934 filed Jan. 11, 1989, now abandoned, all of which are hereby expressly incorporated by reference in their entireties. FIELD OF THE INVENTION [0002] The present invention is related generally to the isolation and characterization of new genes and proteins. More particularly, the present invention is related to providing isolated, substantially pure, biologically active human thyrotropin (hTSH) synthesized by a cloned gene. BACKGROUND OF THE INVENTION [0003] Thyrotropin (TSH) is a pituitary peptide hormone which regulates important body functions. However, heretofore there was no stable, reliable and economic means of synthesizing this important hormone. SUMMARY OF THE INVENTION [0004] It is, therefore, an object of the present invention to provide biologically active, synthetic human thyrotropin in substantially pure, isolated form. [0005] It is another object of the present invention to provide a cloned gene which directs the expression of biologically active human thyrotropin in a suitable vector. [0006] It is a further object of the present invention to provide an assay kit for measuring thyroid-stimulating hormone as well as other thyrotropin substances such as thyroid-stimulating immunoglobulins and the like. [0007] It is a further object of the present invention to provide a method of diagnosing and treating human thyroid cancer. [0008] Other objects and advantages will become evident from the following detailed description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS [0009] These and other objects, features and many of the attendant advantages of the invention will be better understood upon a reading of the following detailed description when considered in connection with the accompanying drawings wherein: [0010] FIG. 1 shows schematic construction of the expression vectors. pSV2.G and pAV2 are pBR322 derived plasmids with the origin of replication (ori) and ampicillin resistance gene (amp r) as shown. pSV2.G contains the early promoter of SV40 upstream of the HindIII cloning site, rabbit .beta.-globin cDNA, and poly-adenylation site/intron of SV40. pAV2 has the entire adenovirus-associated virus genome (4.7 kb) with its three promoters P5, P19, P40, and polyadenylation site. The HindIII cloning site is downstream of the P40 promoter. Human TSH.beta. and hCG.alpha. was inserted into the HindIII site of either plasmid, forming pAV2-hTSH.beta., pAV-hCG.alpha., pSV2.GhTSH.beta., and pSV2.G-hCG.alpha.. [0011] FIG. 2 shows Northern blot analysis of transfected 293 and COS cells. Total cellular RNA was separated on a 1% agarose-formaldehyde gel and transferred to a nylon membrane. Forty micrograms of total RNA from a control of transfected cell culture were applied to each lane. Human CG.alpha. and hTSH.beta. were abbreviated .alpha. and .beta. in construct names and in other figures. Cells that were not transfected are labeled control. Cells transfected with a calcium phosphate precipitate lacking DNA are labeled mock. Lanes 1-4 are total RNAs derived from 293 cells; lanes 5-9 are RNAs derived from COS cells. The migration position of an RNA standard in kilobases and hTSH.beta. mRNA from human pituitary is shown to the left of the autoradiograph. Below the autoradiograph is a simplified version of FIG. 1 showing the pAV2 and pSV2.G plasmid as a single line, the promoters as blackened circles, the 2.0 kb hTSH.beta. genomic fragment as a box containing two exons (blackened regions) and known polyadenylation signal-site sequences as open arrowheads. Below each construct, pAV2-.beta. and pSV2.G-.beta., is the predicted RNA initiating at the specified promoter, and splicing as shown. Solid arrowheads, poly(A) tails. Predicted size in kilobases (kb) is shown to the right of each mRNA species. [0012] FIG. 3 shows the results of gel chromatography. Cell medium from 293 cells transfected with pAV2-hCG.alpha./pAV2-hTSH.beta./pVARNA was chromatographed on a Sephadex G-200 fine column. In addition, standard preparations of hTSH, hCG.alpha., and hTSH.beta. (described in the text) were chromatographed on the same column. RIA of human .alpha. and TSH.beta. and IRMA for hTSH was done on each 1.5-ml fraction. Elution position of bovine thyroglobulin (void, V.sub.o), BSA (67,700 (67 k)) and ovalbumin (45,000 (45 k)) is marked, as well as those of the standard preparations of hTSH, hCG.alpha., and hTSH.beta.. [0013] FIG. 4 shows the results of human TSH IRMA. A highly sensitive and specific hTSH IRMA was performed on two pituitary hTSH standards. World Health Organization 80/558 (WHO STD) and NIH I-6 (1-6 STD), and the medium from 293 cells after transfection with pAV2-hCG.alpha., pAV2-hTSH.beta., and pVARNA (pAV2.alpha./.beta./pVARNA). A logit transformation of assay binding was plotted vs. arbitrary units of sample volume added to the assay. [0014] FIG. 5 shows the results of in vitro bioassay of hTSH in rat thyroid cells. The human pituitary TSH standards and medium from transfected 293 cells used in this assay are defined in the legend to FIG. 4. This in vitro bioassay measures TSH stimulated .sup.125I uptake into rat thyroid cells (FRTL5). Iodide trapping by pituitary standards and medium from a transfected culture are normalized to TSH immunoactivity in an IRMA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT [0015] The above and various other objects and advantages of the present invention are achieved by the cloning of complete nucleotide sequence which directs the synthesis of biologically active human thyrotropin in a suitable expression vector and isolating substantially pure form of the synthesized hormone. [0016] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference. Unless mentioned otherwise, the techniques employed herein are standard methodologies well known to one of ordinary skill in the art. [0017] The term "substantially pure" as used herein means as pure as can be obtained by employing standard conventional purification techniques known in the art. [0018] The term "biologically active" as used herein means that the recombinant hormone, even though not identical in physical or chemical structure or composition as the naturally occurring hormone, yet is functionally equivalent thereto. Continue reading... Full patent description for Biologically active synthetic thyrotropin and cloned gene for producing same Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Biologically active synthetic thyrotropin and cloned gene for producing same patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. 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