结核分枝杆菌抗原HBHA在耻垢分枝杆菌中的重组表达和鉴定

Recombinant Expression and Identification of Antigen Heparin-binding Hemagglutinin(HBHA)of Mycobacterium Tuberculosis in Mycobacterium Smegmatis

目的构建高效表达结核分枝杆菌抗原肝素结合血凝素(HBHA)的重组耻垢分枝杆菌。方法针对结核分枝杆菌H37Rv株HBHA的编码基因Rv0475设计特异引物,利用PCR技术从细菌基因组中获取目的基因,并克隆入pET-30b(+)载体构建重组原核表达质粒pETHBHA,经IPTG诱导pETHBHA转化的重组E.coli BL21(DE3)细菌,Ni柱纯化携带C-端His标签的HBHA蛋白,并制备鼠源多克隆抗体。PCR扩增含自身启动子和调节序列的Rv0475基因并克隆入大肠埃希菌-分枝杆菌穿梭载体pMV261,构建重组分枝杆菌表达质粒pMVHBHA,并经酶切鉴定证实后,电转化入耻垢分枝杆菌,涂布含卡那霉素的7H11平板,挑选抗性克隆并扩大培养,15%SDS-PAGE和Western blot鉴定重组耻垢分枝杆菌的表达。结果分别成功构建重组原核表达质粒pETHBHA和重组分枝杆菌表达质粒pMVHBHA;重组蛋白HBHA在大肠埃希菌中成功表达并纯化后,BCA蛋白浓度测定试剂盒测定浓度为1.912 5μg/μL,用其制备鼠源多抗;证实HBHA利用其自身的启动子和调节序列在重组耻垢分枝杆菌中表达。结论成功构建过表达HBHA的重组耻垢分枝杆菌,为研究该蛋白参与结核病的致病机制和研发新型疫苗和诊断试剂奠定了基础。

Objective To establish recombinant Mycobacterium smegmatis（M.smegmatis）strain highly expressing antigen heparin-binding hemagglutinin（HBHA）of Mycobacterium tuberculosis（M.tuberculosis）.Methods The gene Rv0475,encoding HBHA of M.tuberculosis H37 Rv strain was obtained by PCR and cloned into the vector pET-30b（＋）,to construct the recombinant prokaryotic expression plasmid pETHBHA.Recombinant protein HBHA was prepared as C-terminal His-tagged fusion protein from pETHBHA transformed E.coli BL21（DE3）strain after induction with IPTG and was used to prepare mouse original polyclonal antibodies.Rv0475 containing its promoter and regulatory sequence was amplified by PCR and cloned into E.coli-Mycobacteriumshuttle vector pMV261,namely pMVHBHA.pMVHBHA was transformed into M.smegmatis after identification by restriction enzyme analysis,and recombinant M.smegmatis strain was cultivated on 7H11 plate.The kanamycinresistant clones were identified with 15% SDS-PAGE and Western blotting.Results Recombinant prokaryotic expression plasmid pETHBHA and recombinant mycobacteria expression plasmid pMVHBHA were constructed successfully.The recombinant protein HBHA was successfully expressed and purified in recombinant E.coli strain.The protein concentration was 1.912 5μg/μL determined by a method called BCA Protein Assay Kit,and the protein was used to prepare mouse original polyclonal antibodies.Recombinant protein HBHA was expressed in recombinant M.smegmatis using its own promoter and regulatory sequences.Conclusion Recombinant M.smegmatis strain expressing HBHA was established successfully,which provides foundation to study pathogenesis of tuberculosis and development of new vaccines and diagnostic reagents for TB.