结核分枝杆菌MPT-64分泌蛋白在原核表达载体中的克隆,表达与鉴定

目的　对结核分枝杆菌的一种分泌蛋白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。方法　以结核杆菌H3 7Rv株基因组DNA为模板 ,用PCR法对基因MPT - 6 4进行扩增 ,产物与载体质粒pGEX - 4T - 1构建表达MPT - 6 4的重组质粒 ,将此重组质粒先转化到E coliDH5α内抽提质粒 ,酶切检验 ,再转化入表达宿主E coliBL2 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 ,通过电转移将胶中蛋白转到硝酸纤维素薄膜上后进行免疫染色 ,一抗为结核分枝杆菌菌株H37Rv羊抗阳性血清。结果　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 5 0 0 0 0 ,抗体检测有特异带大小为 5 0kDa。结论　目的基因克隆到菌株内 ,重组结核杆菌分泌蛋白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抗原、抗体的大规模制备打下基础

Cloning,expression and identification of MPT-64 secreted proteins from Mycobacterium tuberculosis

The purpose of this stuties is to clone,identify,and express the secreted protein MPT 64 from Mycobacterial tuberculosis and to play a foundation for diagnosis of TB,for applying TB vaccine into clinic practice use,and for detecting of immunity effectiveness The gene encoding for protein MPT 64 was amplified from M tuberculosis H37Rv genomic DNA by using PCR technique PCR product was cloned initially into the pGEX 4T 1 expression vector,then transformed into E coli DH5αStrain,clones containing the vectors were selected on LB plus ampicillin(100μg/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 50kDa(containing GST) Proteins on the SDS PAGE gel were transferred to nitrocellulose membrane and detected with polyclonal antiserum of M TB H37Rv,specific protein was visualized on gel at molecular mass 50kDa In conclusion,we have obtained recombinant pGEX 4T 1expression vector containing MPT 64 and MPT 64 protein which has been expressed successfully It will establish the detecting of immunity effectiveness and preparation of the antigen and antibody of MPT 64 protein on a large scale