CAJ | 학술논문

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pMVHB‐HA ；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1.9125μg／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HB‐HA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。
목적：구건고효표체결핵분지간균항원간소결합혈응소（HBHA）적중조치구분지간균。방법침대결핵분지간균H37Rv주 HBHA적편마기인Rv0475설계특이인물，이용 PCR기술종세균기인조중획취목적기인，병극륭입pET‐30b（＋）재체구건중조원핵표체질립pETHBHA ，경IPTG유도pETHBHA 전화적중조 E.coli BL21（DE3）세균， Ni주순화휴대C‐단 His표첨적HBHA단백，병제비서원다극륭항체。PCR확증함자신계동자화조절서렬적Rv0475기인병극륭입대장애희균‐분지간균천사재체pMV261，구건중조분지간균표체질립pMVHBHA ，병경매절감정증실후，전전화입치구분지간균，도포함잡나매소적7H11평판，도선항성극륭병확대배양，15％ SDS‐PAGE화Western blot감정중조치구분지간균적표체。결과분별성공구건중조원핵표체질립 pETHBHA 화중조분지간균표체질립pMVHB‐HA ；중조단백 HBHA재대장애희균중성공표체병순화후，BCA단백농도측정시제합측정농도위1.9125μg／μL ，용기제비서원다항；증실HBHA이용기자신적계동자화조절서렬재중조치구분지간균중표체。결론성공구건과표체HB‐HA적중조치구분지간균，위연구해단백삼여결핵병적치병궤제화연발신형역묘화진단시제전정료기출。
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 H37Rv strain was obtained by PCR and cloned into the vector pET‐30b(+ ) ,to construct the recom‐binant prokaryotic expression plasmid pET HBHA.Recombinant protein HBHA was prepared as C‐terminal His‐tagged fusion protein from pET HBHA transformed E.coli BL21(DE3) strain after induction with IPTG and was used to prepare mouse orig‐inal polyclonal antibodies.Rv0475 containing its promoter and regulatory sequence was amplified by PCR and cloned into E.coli‐Mycobacterium shuttle vector pMV261 ,namely pMVHBHA.pMVHBHA was transformed into M.smegmatis after i‐dentification by restriction enzyme analysis ,and recombinant M.smegmatis strain was cultivated on 7H11 plate.The kanamycin‐resistant clones were identified with 15% SDS‐PAGE and Western blotting.Results Recombinant prokaryotic expression plas‐mid 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 anti‐bodies.Recombinant protein HBHA was expressed in recombinant M.smegmatis using its own promoter and regulatory se‐quences.Conclusion Recombinant M.smegmatis strain expressing HBHA was established successfully ,which provides founda‐tion to study pathogenesis of tuberculosis and development of new vaccines and diagnostic reagents for TB.