MTB39A蛋白在昆虫细胞-杆状病毒系统中的表达与鉴定  

Expression and characterization of MTB39A protein in an insect cell-baculovirus system

作　　者：王璞 黄月 姜玲玲 张刚 蔡玉荣 李勇[1] WANG Pu;HUAN Yue;JIANG Lingling;ZHANG Gang;CAI Yurong;LI Yong(School of Life Science,Key lab of Ministry of Education for Protection and Utilization of Special Biological Resources in Western.Yinchuan 750021,China)

机构地区：[1]宁夏大学生命科学学院,宁夏大学西部生物资源保护与利用教育部重点实验室,宁夏银川750021

出　　处：《中国病原生物学杂志》2024年第1期20-24,共5页Journal of Pathogen Biology

基　　金：宁夏自然科学基金项目(No.2022AAC03084);国家自然科学基金区域联合基金项目(No.U22A20525);宁夏回族自治区重点研发计划项目资助课题(No.2021BEF02028);宁夏回族自治区重点研发计划项目资助课题(东西部合作,No.2017BN04)。

摘　　要：目的旨在获得具有免疫原性的结核分枝杆菌(Mycobacterium tuberculosis,MTB)MTB39A蛋白。方法选择H37Rv株中的MTB39A(Rv1196)基因序列(GenBank ID:NC_000962.3)克隆至pFastBac-Dual载体pH启动子下游并进行鉴定。将鉴定正确的重组质粒转化入感受态细胞DH10 Bac中进行蓝白斑筛选,获得的重组杆状质粒Bacmid利用脂质体转染至昆虫细胞Sf9中进行病毒拯救。转染后96 h,收取上清病毒液,盲传3代后采用直接和间接免疫荧光法及Western blot检测MTB39A蛋白的表达。结果重组质粒pFastBac-dual-MTB39A-mCherry和重组Bacmid构建正确,Western blot检测显示MTB39A蛋白能被His-tag抗体识别,IFA鉴定出现特异性红色荧光。结论通过昆虫杆状病毒表达系统成功表达具有反应原性的MTB39A蛋白,为新型结核疫苗候选物和潜在特异性诊断标志物的研制奠定了实验基础。Objective To obtain immunogenic Mycobacterium tuberculosis MTB39A protein.Methods The MTB39A(Rv1196)gene sequence(GenBank ID:NC_000962.3)from the H37Rv strain was selected to be cloned downstream of the pH promoter of the pFastBac-Dual vector and characterized.The correctly identified recombinant plasmid was transformed into receptor cell DH10 Bac for blue-white spot screening,and the obtained recombinant rod-shaped plasmid Bacmid was transfected into insect cell Sf9 for virus rescue using liposomes.At 96 h after transfection,supernatant viral solution was collected,and the expression of MTB39A protein was detected by direct and indirect immunofluorescence and Western blot after 3 generations of blind transmission.Results Recombinant plasmid pFastBac-dual-MTB39A-mCherry and recombinant Bacmid were constructed correctly,and Western blot assay showed that MTB39A protein could be recognized by His-tag antibody,and the IFA identification appeared specific red fluorescence.Conclusion Successful expression of the reactive MTB39A protein by an insect baculovirus expression system lays the experimental foundation for the development of novel tuberculosis vaccine candidates and potential specific diagnostic markers.

关 键 词：结核分枝杆菌 MTB39A蛋白 昆虫细胞-杆状病毒系统 

分 类 号：R378.911[医药卫生—病原生物学]