Cloning Of Muscle-Specific Kinase Gene And Expression In E.Coli

Muscle-specific kinase (MuSK) is a transmembrane protein which is related with acetylcholine receptor, in the development of skeletal muscle, MuSK is a primary ingredient to establish the main synaptic skeleton of the postsynaptic membrane formation. Myasthenia gravis (MG) is a autoimmune disease which caused by the number missing and functional abnormalities of acetylcholine receptor (AChR) at the neuromuscular junction (NMJ) postsynaptic membrane. We found that in a part of AChR-Ab seronegativity MG patients’body there have another antibody MuSK-Ab, but MuSK-Ab in the patients’body were no regularity, its pathogenesis has yet to secure a clear, are still research in.In this study, we obtained the mus musculus muscle-specific kinase gene coding sequences which is numbered AY360453form the gene library, in the premise of does not change the amino acid sequence we change the MuSK gene to E.coli M15preference codes, and compose it. Designed the primers which were added BamHI and KpnI restriction sites, PCR amplified the1.4kb target gene fragment and constructed it in E.coli expression vector pQE30, than we got the recombinat plasmid pQE30-MuSK and transformed into E.coli JM109competent cells, we got the positive clones by colony PCR, restriction enzyme digestion and sequencing. Extraction the recombinat plasmid and transformed into E.coli M15competent cells, induced by the final concentration of0.3mmol/L IPTG, the target protein bands was determined by SDS-PAGE, and use the MuSK Elisa kit tests the immunocompetence of the target protein and its content, the initial expression of MuSK protein was about8.2pmol/L. Used the single factor screening test method to optimize the concentration of IPTG, induction temperature, induction time, carbon/nitrogen source and pH of medium, incubation temperature before induction. The experimental results show that:we successfully constructed the prokaryotic expression vector pQE30-MuSK, and the MuSK protein was expressed in E.coli by the form of inclusion bodies, the relative molecular mass of MuSK protein is about48kDa, the optimum conditions of induced expression were:a final concentration of0.3mmol/L IPTG induction, shaking culture at30℃for12～16h. The best carbon source of medium was sucrose, the best nitrogen source was soybean peptone, the optimum pH was7.0, the optimal incubation temperature before induction was34℃. MuSK proein content was19.74pmol/L after optimized which measured by Elisa Kit, it’s significantly improved compared to the initial expression.