【摘要】 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。更多还原

【Abstract】 Purpose To clone and express the ag85b ,one gene of the mycobacterium secreted proteins,and to establish a basis for diagnasis of tuberculosis. Methods We used genomic DNA of M.tuberculosis H37Rv strain as template,amplified the ag85b gene with PCR constructed the recombinant plasmid with ag85b and vector plasmid pET24b,transformed the recombinant plasmid pET24b ag85b into E.coli DH5α.Then purified the plasmid DNA,tested it by restriction endonucleases digest,transformed the plasmid into expressive strain E.coli JM109(DE3) again,induced the transformants with IPTG,split bacteria,centrifuged the split fliud and the supernatant was used for electrophoresis. Results SDS?PAGE demonstrated that the transformants expressed Ag85B,the protein molecular weight was 30 000. Conclusions The target gene has been cloned into host bacterium strain.Expression of Ag85B makes a basis of diagnosis of tuberculosis.更多还原