南方医科大学学报

南方医科大学学报 ›› 2012, Vol. 32 ›› Issue (12): 1713-.

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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

宋远斌，何思杰，余楠，陈欣欣，王斌，车小燕，曾其毅   

  • 出版日期:2012-12-20 发布日期:2012-12-20

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

  • Online:2012-12-20 Published:2012-12-20

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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8mol/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。

Abstract: ObjectiveTo clone and express VP1-VP4genes encoding the structural proteins of Coxsackie virus A16and analyze
the antigenicity of the expressed recombinant proteins.MethodsThe VP1-VP4cDNAs were amplified with RT-PCR from the
extracted viral RNA and cloned into pMD19-T vectors. The VP1-VP4genes were inserted to the multi-cloning sites of the
plasmid pQE30a, and the protein expressions inE. coliM15were induced by IPTG. After purification by washing with8mol/L
urea under denaturing condition, the recombinant proteins were identified by Western blotting and ELISA for their
immunogenicity against rabbit antisera of Coxsackie virus A16and enterovirus71, respectively. ResultsThe recombinant
VP1-VP4proteins were highly expressed inE. coliM15. The purified proteins could be recognized by rabbit antiserum of
Coxsackie virus A16and showed cross reactivity with the rabbit antiserum of Enterovirus71. ConclusionThe recombinant
Coxsackie virus A16VP1-VP4proteins obtained possess good antigenicity.