Cloning And Expression Of E.coli Preferred Coding DNA Sequence Of Human Thyroid-stimulating Hormone β Chain

Thyrotropin (thyroid-stimulating hormone, TSH) is a glycoprotein(MW 28,000) composed of two noncovalently linked alpha and beta subunit.The structure of the alpha subunit of TSH is identical to that of the otherglycoprotein molecules-FSH, LH, and human chorionic gonadotropin (hCG)-but the beta subunit differs in these glycoproteins and is responsible for theirbiologic and immunologic specificity. TSH attaches to high-affinity receptors inthe thyroid (TSH receptor), stimulating iodide uptake, hormonogenesis, andrelease of T4 and T3.TSH secretion also causes an increase in gland size andvascularity by promoting mRNA and protein synthesis. In Graves'disease, Tlymphocytes become sensitized to antigens within the thyroid gland andstimulate B lymphocytes to synthesize antibodies to these antigens. One suchantibody directly acts on the TSH receptor site in the thyroid cell membrane andhas the capacity to stimulate the thyoid cell to increased growth andfunction. The presence of this circulating antibody is positively correlated withactive disease and with relapse of the disease. According to theIdiotype-antiidiotype immunologic network theory, the antibody is considered asthe Ab2βof TSH-the "internal image" of TSH. To obtain enough TSHβfor basicand clinic research, the recombinant human TSHβwas expressed in E. coli.In this research, the codons in the native human TSHβcDNA werereplaced by those E.coli preferred while maintaining the amino acid sequence.The modified coding sequence was synthesized chemically in vitro. And thenthe target sequence was inserted into prokaryotic fusion expression vectorpGEX-3X and pQE-30. Finally the recombinant plasmid pGEX-3X/hTSHβandpQE-30/hTSHβwere transformed respectively into E.coli DH5αand E. coli M 15[pREP4] for expression.SDS-PAGE and Western Blotting showed that The GSTfusion protein was about 42KD and mainly existed in inclusion bodies. Theproduction of rhTSHβcould account for 26.57% of the total sediment protein ofE.coli DH5αwith optimizing the culture conditions. After solubilization and extraction, the rhTSHβwas renaturated by stepwise diluting and wasdialyzed.The purity of the rhTSHβcould be 82% by electroelution. The purifiedrhTSHβwas analysed by Electrochemiluminescence immunoassay.