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目的克隆结核分枝杆菌Ag85B基因,在大肠埃希菌中进行表达,获得纯化的重组Ag85B蛋白。方法以结核分枝杆菌H37Rv基因组DNA为模板,应用PCR方法扩增Ag85B基因,以质粒PET23a(+)为表达载体构建Ag85B重组质粒,转化大肠埃希菌BL21(DE3),以IPTG诱导表达,通过SDS-PAGE电泳和Western blot技术分析鉴定Ag85B蛋白表达,采用Novagen公司生产的HisBind蛋白纯化试剂盒纯化Ag85B蛋白。结果构建了具有正确基因序列的Ag85B重组表达质粒,经SDS-PAGE分析,诱导表达的重组蛋白分子质量单位约为33ku,Western blot鉴定能被抗His单抗识别,该重组Ag85B蛋白在大肠埃希菌BL21(DE3)中以包涵体形式存在。结论目的基因克隆入宿主菌中并表达成功,纯化的重组Ag85B蛋白为进一步的研究与应用奠定了基础。
Objective To clone Mycobacterium tuberculosis Ag85B gene and express it in Escherichia coli to obtain purified recombinant Ag85B protein. Methods The genomic DNA of Mycobacterium tuberculosis H37Rv was used as template to amplify Ag85B gene by PCR. Ag85B recombinant plasmid was constructed by using plasmid pET23a (+) as an expression vector. The recombinant plasmid was transformed into E. coli BL21 (DE3) and induced by IPTG. Ag85B protein was identified by SDS-PAGE electrophoresis and Western blot analysis. Ag85B protein was purified using HisBind protein purification kit from Novagen. Results Ag85B recombinant plasmid with the correct gene sequence was constructed. The molecular weight of the recombinant protein induced by SDS-PAGE analysis was about 33ku. The recombinant protein was identified by Western blot using anti-His monoclonal antibody. The recombinant Ag85B protein was expressed in E. coli The bacteria BL21 (DE3) exist as inclusion bodies. Conclusion The target gene was cloned into host bacteria and expressed successfully. The purified recombinant Ag85B protein laid the foundation for further research and application.