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目的 对分支杆菌的一种分泌蛋白Ag85B的基因进行克隆、表达 ,为结核病进行诊断打下基础。 方法 以结核杆菌H37Rv株基因组DNA为模板 ,以PCR法对基因ag85b进行扩增 ,产物与载体质粒 pET2 4b构建表达Ag85B的重组质粒 ,将此重组质粒先转化入大肠杆菌DH5α内 ,抽提质粒 ,酶切检验 ,再转化入表达宿主大肠杆菌JM 1 0 9(DE3)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。结果 电泳发现转化了重组质粒的菌株有蛋白表达 ,所表达的蛋白质分相对分子质量为 30 0 0 0。结论 目的基因克隆入宿主菌内 ,重组结核杆菌分泌蛋白Ag85B的成功表达为进行临床诊断试验奠定了基础。
Objective To clone and express a secreted protein Ag85B from Mycobacterium tuberculosis and lay the foundation for the diagnosis of tuberculosis. Methods The genomic DNA of Mycobacterium tuberculosis H37Rv strain was used as a template to amplify the gene ag85b by PCR. The recombinant plasmid pET2 4b expressing Ag85B was constructed. The recombinant plasmids were first transformed into E. coli DH5α, Digested with enzyme digestion, and then transformed into E. coli JM109 (DE3) host strain. After induced by IPTG, the transformed strain was broken, centrifuged and the supernatant was subjected to SDS PAGE. Results The results of electrophoresis showed that the recombinant plasmids transformed with the recombinant protein had protein expression, and the relative molecular mass of the expressed protein was 30 000. Conclusion The target gene was cloned into the host bacterium, and the successful expression of recombinant Mycobacterium tuberculosis secreted protein Ag85B laid the foundation for clinical diagnosis.