人心肌肌钙蛋白I基因的人工合成及高效表达  

Cloning and overexpression of human cardiac troponin I gene

作　　者：徐霞[1] 杨能[1] 涂洪斌[2] 

机构地区：[1]广州医学院检验系 [2]广州医学院实验医学研究中心,广州510182

出　　处：《免疫学杂志》2005年第4期320-322,326,共4页Immunological Journal

基　　金：广东省医学科学技术研究基金资助项目(A2000265)

摘　　要：目的人工设计合成完整的人心肌肌钙蛋白I(hcTnI)基因,克隆并在大肠杆菌中获得高效表达。方法根据大肠杆菌遗传密码的偏爱性,人工设计合成hcTnI基因,测序正确后通过DNA重组技术将其插入温控型表达载体pBV220,转化大肠杆菌(E.coli)DH5α,构建cTnI基因的非融合表达菌hcTnI-pBV220/DH5α,热激后诱导非融合蛋白的表达,用单克隆抗体检测特异性hcTnI蛋白表达。结果序列分析表明克隆载体中的cTnI人工基因与设计相符,双酶切电泳结果证明表达载体构建成功,经SDS-PAGE证实重组蛋白的相对分子质量(Mr)约24000,凝胶密度扫描约占菌体总蛋白的25%,能与cTnI单克隆抗体特异性结合。结论成功获得cTnI人工基因,构建了cTnI基因原核表达载体并获得了高效表达,为制备高特异性的抗体及临床检测应用奠定了基础。Objective To design and clone full human cardiac troponin I (hcTnI) gene and construct an efficient expression system for hcTnI in prokaryotic cells. Methods According to the codon usage bias of E.coli, an artificial gene was designed for cTnI. The gene was synthesized, ligated by PCR, and then cloned into a prokaryotic nonfusion-protein type vector pBV220 and subsequently transformed into E.(coli) DH5α, so as to construct prokaryotic nonfusion-protein expression bacterial hcTnI- pBV220/DH5α. After heat shock, the hcTnI nonfusionprotein was induced to express, and then identified by SDS-PAGE. The expressed hcTnI nonfusion protein was the specific antigen against anti-(cTnI) monoclonal antibodies. Results The nucleotide sequence analysis indicated that the sequence of cloned artificial cTnI gene was accorded with that of designed. The positive clones were primarily confirmed by anti-ampicillin selection and restrictive enzyme digestion. The relative molecular weight of hcTnI nonfusionprotein was about M_r 24 000 identified by SDS-PAGE The active human cTnI protein was obtained and amounted to 25 % of total bacterial protein. Conclusion The human cTnI artificial gene is obtained and successfully constructed its prokaryo-(tic) expression vector, which will provide theoretical basis for preparation of high-specific monoclonal antibody and clinical laboratory diagosis of cTnI.

关 键 词：肌钙蛋白Ⅰ 密码子偏爱性 克隆 表达 

分 类 号：Q78[生物学—分子生物学]