Expression Of The Extracellular Coding Region Of Human Acetylcholine Receptor Alpha Subunit And Its Gene Immune

Human Acetylcholine receptor a-subunit is the main immunogenic region of the autoimmune disease-myasthenia gravis. The structure and function of auto-antigen is a bottleneck for breaking through the mechanism of autoimmune diseases and the possible therapeutic methods. It is obviously hard to get the enough material from natural source for the research and therapy. It is undoubted that an ideal choice to obtain sufficient amounts of the protein is performed by using gene engineering technology.(1)The gene fragment of human acetylcholine receptor a- subunit N-terminal 211 residues (AChR_α211) was cloned to various vectors: to be four recombinant plasmids, pUC-AChR_α205 , pET- AChR_α211 , pUC-CBD-AChR_α211 , pET- CBD-AChR_α211. After transformation, the recombinant strains were cultured and induced in the definite conditions. It was shown from experimental results that the target protein expressed in E.coli JM109/pUC-AChR_α205 was soluble, while others existed in the form of inclusion bodies. After denaturation and renaturation of inclusion bodies expressed from the strain E.coli BL21 (DE3)/pET-CBD-AChR_α211, the refolded protein CBD-AChR_α211 can be cleaved with protease thrombin, forming two parts of AChR_α211 (25 kDa) and CBD(5 kDa). It was also shown from Western blot analysis that both CBD-AChR_α211 and AChR_α211 had the very similar immunoreactivity, compared with AChR a-subunit from Torpedo.(2)Gene vaccine pcDNA-AChR_α211 was prepared by inserting the gene fragment of human acetylcholine receptor a-subunit N-terminal 211 residues (AChR_α211) into shuttle plasmid pcDNA3.0 and directly injected intramuscularly into C57BL/6 mice. After immunization, the corresponding...