结核分枝杆菌16kD-CFP10-19kD融合蛋白的构建表达及纯化

Construction, Expression and Purification of Recombinant Fusion Proteins 16kD-CFP10-19kD of Mycobacterium tuberculosis

目的构建结核分枝杆菌(MTB)16k D-CFP10-19k D融合蛋白表达载体,在大肠杆菌中对蛋白进行表达并经过纯化获得16k D-CFP10-19k D融合蛋白。方法利用聚合酶链式反应(PCR)方法以结核分枝杆菌H37Rv基因组为模板扩增16k D、CFP10及19k D基因,经过限制性内切酶处理并依次与p ET28a载体连接,构建p ET28a-16k D-CFP10-19k D重组质粒,转化至大肠杆菌表达株BL21中,在IPTG及低温诱导下获得目标蛋白,镍柱层析获得纯化蛋白。结果经过酶切及测序鉴定验证p ET28a-16k D-CFP10-19k D重组质粒构建正确,原核表达获得可溶性目标蛋白并对其初步纯化。结论成功构建表达并纯化获得16k D-CFP10-19k D融合蛋白,为开发新的结核病特异性诊断抗原研究奠定基础。

Objective 16kD-CFP10-19kD fusion protein expression vector was constructed and expressed in Eseherichia coli and purified to obtain 16kD-CFP10-19kD fusion protein. Methods 16kD, CFP10 and 19kD genes were amplified by polymerase chain reaction from Mycobacterium tuberculosis H37Rv genome. The gene fragment was treated with restriction enzyme and inserted into pET28a vector in order to construct the recombinant plasmid of pET28a-16kD, CFP10, 9kD. The recombinant plasmid was transformed into Escherichia coli strain BL21, and the target protein was obtained by IPTG under low temperature. Results The recombinant plasmid of pET28a-16kD-CFP10-19kD was constructed correctly by enzyme digestion and sequencing. The soluble protein was obtained by prokaryotic expression and preliminary purification. Conclusion Construction, expression and purification of 16kD-CFP10-19kD fusion protein, as the basis for the development of new diagnostic antigens of new tuberculosis.