猪带绦虫14-3-3.2原核表达系统的构建及其在猪带绦虫成虫和囊尾蚴中的表达  

Construction of prokaryotic expression system of Taenia solium 14-3-3.2 and its expression at the stages of Taenia solium adult and cysticercus

作　　者：刘美辰 欧阳任辉 罗波 周必英 Liu Meichen;Ouyang Renhui;Luo Bo;Zhou Biying(Department of Parasitology,Zunyi Medical University,Zunyi 563000,China;Department of Medical Laboratory,the Second People's Hospital of Guizhou Province,Guiyang 550000,China)

机构地区：[1]遵义医科大学寄生虫学教研室,贵州遵义563000 [2]贵州省第二人民医院医学检验科,贵阳550000

出　　处：《中华地方病学杂志》2021年第6期435-440,共6页Chinese Journal of Endemiology

基　　金：贵州省科技计划项目(黔科合基础[2018]1190);贵州省教育厅青年科技人才成长项目(黔教合[2018]225);省市科技合作专项资金项目(省市科合[2015]52)。

摘　　要：目的建立猪带绦虫(Ts)14-3-3.2原核表达系统,并观察Ts14-3-3.2蛋白在Ts成虫和囊尾蚴中的表达情况。方法在遵义医科大学寄生虫学教研室前期获得Ts14-3-3.2基因序列的基础上,采用基于PCR的精确合成(PAS)方法全基因合成Ts14-3-3.2基因,经限制性内切酶NdeⅠ和XbaⅠ双酶切后连接质粒pCzn1,构建重组质粒pCzn1-Ts14-3-3.2。将重组质粒转化至大肠埃希菌ArcticExpress感受态细胞中诱导表达,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和考马斯亮蓝染色分析和鉴定表达产物,通过镍(Ni)柱亲和纯化获得纯化Ts14-3-3.2重组蛋白。采用该纯化重组蛋白免疫新西兰大白兔,制备Ts14-3-3.2重组蛋白多克隆抗体,采用免疫印迹法(Western blot)检测Ts14-3-3.2蛋白在Ts成虫和囊尾蚴中的表达情况。结果成功构建重组质粒pCzn1-Ts14-3-3.2,诱导表达后菌体上清液和沉淀均在相对分子质量约29.31×10^(3)处出现Ts14-3-3.2目的蛋白条带。纯化后带有His标签的Ts14-3-3.2重组蛋白能被抗His单克隆抗体识别,获得效价为1∶512000的Ts14-3-3.2重组蛋白多克隆抗体。Western blot结果显示,Ts14-3-3.2蛋白在Ts成虫和囊尾蚴中均有表达。结论成功建立Ts14-3-3.2原核表达系统,获得了高纯度、高效价的Ts14-3-3.2重组蛋白多克隆抗体。Ts14-3-3.2蛋白在Ts成虫和囊尾蚴阶段均有表达。Objective To establish the prokaryotic expression system of Taenia solium(Ts)14-3-3.2,and observe the expression of Ts14-3-3.2 protein at the stages of Ts adult and cysticercus.Methods Based on the Ts14-3-3.2 gene sequence obtained by the Department of Parasitology,Zunyi Medical University in the previous study,the whole gene was synthesized by PCR-based accurate synthesis(PAS)method.After double digestion with restriction enzymes NdeⅠand XbaⅠ,the plasmid pCzn1 was ligated to construct a recombinant plasmid pCzn1-Ts14-3-3.2.Then it was transformed into Escherichia coli ArcticExpress competent cells to induce the expression of Ts14-3-3.2 protein.The expression products were analyzed and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and coomassie blue staining.The purified Ts14-3-3.2 recombinant protein was obtained by Ni-affinity chromatography.New Zealand rabbits were immunized with the recombinant protein to produce Ts14-3-3.2 polyclonal antibody.Western blotting was used to detect the expression of Ts14-3-3.2 protein at the stages of Ts adult and cysticercus.Results The recombinant plasmid pCzn1-Ts14-3-3.2 was successfully constructed.After induced expression,Ts14-3-3.2 target protein bands appeared in the supernatant and precipitated at the relative molecular weight of about 29.31×10^(3).The purified Ts14-3-3.2 recombinant protein with His label could be recognized by anti-His monoclonal antibody,and the Ts14-3-3.2 polyclonal antibody with titer of 1∶512000 was obtained.Western blotting showed that Ts14-3-3.2 protein was expressed at the stages of Ts adult and cysticercus.Conclusions The prokaryotic expression system of Ts14-3-3.2 is successfully established,and the Ts14-3-3.2 polyclonal antibody with relatively higher purity and titer is obtained.The Ts14-3-3.2 protein is expressed at the stages of Ts adult and cysticercus.

关 键 词：猪带绦虫 囊尾蚴 猪带绦虫14-3-3.2蛋白 表达 

分 类 号：R383.3[医药卫生—医学寄生虫学]