肠道病毒71型VP1～VP4基因克隆及其表达产物的免疫原性  

Gene cloning and immunogenicity analysis of the structural proteins VP1-VP4 of enterovirus 71

作　　者：宋远斌[1] 余楠[2] 何思杰[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]南方医科大学医学检验中心,广东广州510282

出　　处：《南方医科大学学报》2011年第11期1846-1850,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)

摘　　要：目的克隆并表达肠道病毒71型VP1~VP4蛋白基因,初步鉴定其免疫原性。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,表达产物用8 mol/L尿素洗涤及Ni柱亲和层析纯化后,用肠道病毒71型免疫兔血清和柯萨奇病毒A16型免疫兔血清对重组蛋白进行Western blotting及ELISA鉴定。结果构建的重组质粒pQE30a/VP1~VP4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可以被免疫兔血清特异识别。结论肠道病毒71型VP1~VP4蛋白表达载体在大肠杆菌M15中高效表达。纯化产物具有较强的免疫原性,为今后EV71亚单位疫苗的研究和检测试剂盒提供了参考。Objective To clone the genes encoding the structural proteins VP1-VP4 of enterovirus 71 and investigate the immunogenicity of the expressed recombinant proteins.Methods The VP1-VP4 cDNAs were amplified by RT-PCR from the extracted viral RNA and cloned into pMD19-T vector.The cloned VP1-VP4 genes were then inserted into the multi-cloning sites of plasmid pQE30a and expressed in E.coli M15 with IPTG induction.After washing with 8 mol/L urea and purification with Ni-affinity chromatography,the recombinant proteins obtained were tested for immunogenicity by Western blotting and ELISA using rabbit antisera against enterovirus 71 and Coxsackie Virus A16.Results The recombinant VP1-VP4 proteins were highly expressed in E.coli M15 and the purified proteins could be specifically recognized by the rabbit sera against enterovirus 71.Conclusion The expressed enterovirus 71 structural proteins show good immunogenicity and can be used for developing enterovirus 71 vaccine and detection kits.

关 键 词：肠道病毒71型 基因克隆 原核表达 免疫原性 

分 类 号：R392.1[医药卫生—免疫学]