柯萨奇病毒A16型VP1-VP4基因克隆及其表达产物的抗原相关性分析  

Cloning,expression and antigenic analysis of VP1-VP4 gene encoding the structural protein of Coxsackie virus A16

作　　者：宋远斌[1] 何思杰[2] 余楠[2] 陈欣欣[1] 王斌[1] 车小燕[2] 曾其毅[1] 

机构地区：[1]南方医科大学珠江医院儿科中心,广东广州510282 [2]南方医科大学珠江医院医学检验中心,广东广州510282

出　　处：《南方医科大学学报》2012年第12期1713-1717,共5页Journal of Southern Medical University

基　　金：国家重大科技专项课题(2009ZX10004-306);广州市医药卫生科技重大项目(201102A211007);广东省科技计划项目(2010B031600238);广州市计划项目科技支撑计划(2010J-E421)~~

摘　　要：目的克隆并表达柯萨奇病毒A16型VP1-VP4蛋白基因,初步分析其抗原相关性。方法抽提病毒RNA,经RT-PCR方法分别扩增出VP1-VP4蛋白基因片段,经克隆后,在QIA表达系统中表达,表达产物经8 mol/L尿素洗涤及镍柱亲和层析纯化后,用柯萨奇病毒A16型病毒免疫兔血清及肠道病毒71型病毒免疫兔血清对重组蛋白进行Western blotting及ELISA分析其抗原相关性及交叉反应性。结果成功构建的重组质粒pQE30a/VP1-VP4并经IPTG诱导,重组蛋白VP1-VP4高效表达并纯化成功,经Western blotting及ELISA证实重组蛋白VP1-VP4可以被CVA16免疫兔血清特异识别,部分与肠道病毒71型免疫血清存在交叉反应。结论在大肠杆菌M15中高效表达出柯萨奇病毒A16型VP1-VP4蛋白,经纯化产物具有较强的抗原反应性。Objective To clone and express VP1-VP4 genes encoding the structural proteins of Coxsackie virus A16 and analyze the antigenicity of the expressed recombinant proteins. Methods The VP1-VP4 cDNAs were amplified with RT-PCR from the extracted viral RNA and cloned into pMD19-T vectors. The VP1-VP4 genes were inserted to the multi-cloning sites of the plasmid pQE30a, and the protein expressions in E. coli M15 were induced by IPTG. After purification by washing with 8 mol/L urea under denaturing condition, the recombinant proteins were identified by Western blotting and ELISA for their immunogenicity against rabbit antisera of Coxsackie virus A16 and enterovirus 71, respectively. Results The recombinant VP1-VP4 proteins were highly expressed in E. coli M15. The purified proteins could be recognized by rabbit antiserum of Coxsackie virus A16 and showed cross reactivity with the rabbit antiserum of Enterovirus 71. Conclusion The recombinant Coxsackie virus A16 VP1-VP4 proteins obtained possess good antigenicity.

关 键 词：柯萨奇病毒A16型 VP1蛋白 VP2蛋白 VP3蛋白 VP4蛋白 克隆 原核表达 抗原反应性 

分 类 号：R392.1[医药卫生—免疫学]