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目的:通过构建结核分枝杆菌(MTB)16KD蛋白的表达载体,在大肠杆菌(E.coli)中表达MTB16KD蛋白,并获得大量纯化的16KD蛋白。方法:采用聚合酶链反应(PCR)从结核分枝杆菌H37Rv基因组中扩增出16KD蛋白的编码基因,用限制性内切酶消化后插入载体pGEX-4T-2中,测序结果证实后,将重组质粒转化E.coli DH5α,经IPTG诱导表达GST-16融合蛋白。聚丙烯酰胺凝胶电泳(SDS-PAGE)分析融合蛋白的相对分子质量及表达形式。Western blot鉴定16KD蛋白活性。经GST亲和层析柱过柱,得到纯化的16KD蛋白。结果:构建了具有正确基因序列的表达载体,在E.coli DH5α中诱导表达的融合蛋白GST-16占菌体总蛋白的42%。Wstern Blot结果证明:融合蛋白GST-16能与抗结核分枝杆菌的抗体发生特异性结合。紫外分光光度仪测量纯化16KD蛋白的浓度为688mg/L。结论:结核分枝杆菌16KD蛋白在E.coli DH5α能高效表达,该蛋白具有特异的免疫学活性。
OBJECTIVE: To express MTB16KD protein in E. coli and to obtain a large amount of purified 16KD protein by constructing an expression vector of 16KD protein of M. tuberculosis. Methods: The 16KD protein was amplified from Mycobacterium tuberculosis H37Rv by polymerase chain reaction (PCR) and inserted into vector pGEX-4T-2 after digestion with restriction enzyme. After confirming the sequencing results, The recombinant plasmid was transformed into E. coli DH5α, induced by IPTG expression of GST-16 fusion protein. Polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the relative molecular mass of the fusion protein and expression. Western blot identification 16KD protein activity. After the column by GST affinity chromatography to obtain a purified 16KD protein. Results: The expression vector with the correct gene sequence was constructed. The fusion protein GST-16 induced in E. coli DH5α accounted for 42% of the total bacterial proteins. Wstern Blot results show that the fusion protein GST-16 can specifically bind to anti-MTB antibodies. UV spectrophotometer to measure the concentration of purified 16KD protein 688mg / L. Conclusion: Mycobacterium tuberculosis 16KD protein is highly expressed in E. coli DH5α, and the protein has specific immunological activity.