霍乱弧菌CTB基因的克隆、表达及重组蛋白活性分析

Cloning and Expression of Vibrio cholerae CTB Gene and the Recombinant CTB Protein Activation Assay

霍乱弧菌CTB蛋白具有免疫佐剂活性。根据已发表CTB基因的序列设计一对引物，从一株霍乱弧菌中扩增出CTB基因，测序后发现该基因全长375bp，与国内分离的六株CTB基因的同源性达96．0％-99．2％。将该基因与pTWIN1连接构建了原核表达载体pTWIN1-CTB，重组表达载体转化BL21（DE3）表达菌株，0．8mmol／LIPTG诱导4h后，收获的细菌总蛋白SDS—PAGE电泳显示CTB在原核表达系统中得到表达，融合蛋白大小与理论值符合，蛋白产量占细菌总蛋白的20％左右，主要以包涵体形式存在，Western杂交和GM1-ELISA结果表明重组蛋白具有免疫原性和粘膜佐剂活性。

CTB protein possessed mucosal adjuvant immunoactivity. The CTB gene was amplified by PCR method from a strain V. cholerae. The nucleotide sequence of CTB gene was 375 bp and shared 96.0% -99.2% homology with other 6 CTB genes. The recombinant plasmid pTWIN1-CTB transformed E. coli strain BI21 （DE3） expressed with 0. 8 mmol/L IPTG. The molecular weight of expression products was identical with expectative weight by SDS-PAGE electrophoresis. The CTB fusion proteins mainly assembled inclusion bodies and the outputs of proteins were approximately 20% of the total bacterial proteins. The CTB proteins possessed mucosal immunoactivity by GM1-ELISA assay.