结核分枝杆菌候选抗原基因克隆与表达质粒构建

目的 克隆并构建编码结核分枝杆菌(MTB)ESAT6，Ag85B和MPT64分泌蛋白的重组表达质粒。方法 采用聚合酶链反应(PCR)方法从结核分枝杆菌H37Rv基因组DNA中分别扩增出ESAT6，Ag85B和MPT64基因(288bp，978bp and 687bp)，并克隆到T载体，然后用双内切酶消化后，与同样酶消化的pGEX-4T-2连接，转化大肠杆菌E．coli Dh5α，阳性克隆用酶切和DNA测序鉴定。结果 酶切鉴定所切下的片段大小与预计相符，测序结果与献报道一致，证实符合表达框架。结论 成功地克隆并构建了ESAT6，Ag85B和MPT64基因的重组表达质粒pGEX-ESAT6,pGEX-Ag85B和pGEX-MPT64。

Construction of the Recombinant Expression Plasmid Encoding ESAT6 and Ag85B and MPT64 Gene of the Tuberculosis Bacterium

Objective To clone the ESAT6, Ag85B and MPT64 genes of tuberculosis bacterium and construct expression plasmids containing these genes. Methods The ESAT6, Ag85B and MPT64 gene of tuberculosis bacterium were amplified (PCR method) from the H37Rv. The three gene fragments of 288bp, 978bp and 687bp, were cloned into pGEM-T-easy vector and then subcloned into pGEX-4T-2 individually according to its special orientadon. The constructed recombinant plasmids were transformed into? coli DH5a. The positive clones were identified by restriction analysis and DNA sequencing. Results The restriction enzyme digestion patents and the predicted amino acids sequences of the three cloned genes were the same as pervious reports. Conclusion The ESAT6, Ag85B and MPT64 gene were successfully amplified and cloned into pGEX-4T-2 expression vector.