结核分枝杆菌三种分泌蛋白在原核表达载体中的克隆、表达与鉴定

The Cloning,Expression and Identifcation of Ag85B,ESAT-6,MPT-64 Secreted Proteins from Mycobacterial tuberculosis

对结核分枝杆菌的三种分泌蛋白Ag85B ,ESAT_6和MPT_6 4基因进行克隆、鉴定与表达 ,为结核病诊断、重组疫苗应用和免疫效应检测打下基础。以结核杆菌H37RV 株基因组DNA为模板 ,用PCR法对基因Ag85B、ESAT_6、MPT_6 4进行扩增 ,产物与载体质粒pET2 2b和pGEX_4T_1构建表达Ag85B、ESAT_6、MPT_6 4的重组质粒 ,将此重组质粒先转化到大肠杆菌DH5a内抽提质粒 ,酶切检验 ,再转化入表达宿主大肠杆菌BL2 1(DE3)PLysS菌株内 ,对转化菌株以IPTG进行诱导后 ,破菌 ,离心 ,上清进行SDS_PAGE电泳 ,电泳发现转化了重组质粒的菌株有表达蛋白 ,所表达的蛋白质相对分子质量为 30 0 0 0、6 0 0 0、5 0 0 0 0。结果表明 :目的基因克隆到菌株内 ,重组结核杆菌分泌蛋白Ag85B、ESAT_6、MPT_6 4的成功表达为结核病诊断、重组疫苗应用和免疫效应检测以及上述三种抗原。

The purpose of this study is to clone,identify,and express the secreted protein Ag85B,ESAT-6,MPT-64 from Mycobacterial tuberculosis and to play a foundation for diagnosis of TB,for applying TB vaccine into clinic practice,and for detecting of immunity effectiveness.The gene encoding for protein Ag85B,ESAT-6,MPT-64 was amplified from M.tuberculosis H37Rv chromosomal DNA by using PCR technique.PCR product was cloned initially into pET22b and pGEX-4T-l expression vector,then transformed into E.coli. DH5a Strain,Clones containing the vectors were selected on LB-plus ampicillin(100ug/ml)plates,and plasmid DNA was extracted and digested with enzymes.Plasmids containing the right insertion were sequenced to confirm its identity and retransformed the combinants into E.coli. BL21(DE3)PlysS Strain.Bacterial lysates prepared from 1 mmol/L IPTG induced cultures were loaded directly onto SDS-PAGE.Upon IPTG induction,the recombinant pGEX-4T-1-MPT-64 produced indeed a new protein with an apparent MW of 30KDa(containing GST)and the recombinant pET22b-Ag85B,pET22b-ESAT-6 produced proteins with apparent MW of 30KDa and 6KDa respectively.In conclusion,we obtained recombinant pGEX-4T-1 expression vector containing MPT-64 and recombinant pET22b expression vector containing-Ag85B,ESAT-6.Three proteins have been expressed successfully.It will establish the detecting of immunity effectiveness and preparation of the antigen and antibody of three proteins on a large scale.