结核分支杆菌MPT64基因的克隆及其在大肠杆菌中的表达、纯化和诊断运用

目的利用大肠杆菌表达系统大量获得重组MPT64蛋白,分析其生物学、免疫学特性,并初步评估其在结核病血清学诊断方面的价值.方法用聚合酶链反应(PCR)方法从结核分支杆菌H37Rv中获得了MPT64基因,并构建大肠杆菌表达株;聚丙烯酰胺凝胶电泳重组蛋白;Western印迹及酶联免疫吸附测定法(ELISA)分析重组蛋白免疫原性,并检测血清的抗结核抗体.结果构建了能表达重组MPT64的大肠杆菌工程菌,表达的重组蛋白蛋白为可溶性形式,表达量占细胞总蛋白的30%,分子量约23kD.经离子交换柱纯化后,重组蛋白纯度达90%以上.Western印迹结果证实该重组蛋白能与抗结核分支杆菌多克隆抗体发生特异免疫结合反应.ELISA分析表明该纯化的重组抗原能区别抗结核抗体阳性患者血清及正常人血清.结论该实验获得了能高效表达MPT64蛋白抗原的大肠杆菌工程菌.纯化的该重组蛋白具有特异的免疫原性,有一定的诊断运用价值.

Expression of mycobacterium tuberculosis MPT64 in E.coli,purification and application to sera diagnose

Objective:To gain the recombinant MPT64protein of Mycobacterium tuberculosis,study its immuno-logical characteristics and to evaluate its potential value for TB serodiagnosis.Methods:DNA fragments code for the protein were obtained by PCR,then cloned into the expression vector pET-9d to gain recombinant E.coli BL21(DE3).The expressed protein analyzed by SDS-PAGE,and purified by two anion exchange columns.The immuno-logical characteristics of the recombinant MPT64were analyzed by Western-blotting and its serodiagnostic value de-tected by ELISA.Results:The clone was found to produce23kDa protein similar to the molecular weight of natural MPT64Protein.Analyzed by SDS-PAGE,the recombinant MPT64protein existed in the form of solution in E.coli.and amounted to30%of total cell proteins.ELISA results indicated that the purified recombinant protein could dis-tinguish sera from tuberculosis patients with anti-PPD antibody and those from tuberculin positive contacts.Con-clusions:We get recombinant MPT64protein highly expressed in E.coli.This recombinant MPT64showes specific immunogenicity and can be a new serodiagnostic reagent.