人肠道病毒71型及柯萨奇病毒A组16型VP1蛋白的真核表达  

Expression of VP1 from enterovirus 71 and coxsackievirus A16

作　　者：徐晓园 李文丽 徐岚[1] 李蕊[2] XU Xiaoyuan;LI Wenli;XU Lan;LI Rui(Department of Obstetrics and Gynecology,the First Affiliated Hospital of Shantou University Medical College,Shantou 515041;Department of Microbiology and Immunology,Shantou University Medical College,Shantou 515041,Guangdong,China)

机构地区：[1]汕头大学医学院第一附属医院妇产科,广东汕头515041 [2]汕头大学医学院微生物学与免疫学教研室,广东汕头515041

出　　处：《癌变．畸变．突变》2020年第1期43-46,51,共5页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：国家自然科学基金项目(31300761);广东省自然科学基金项目(2016A030310075)。

摘　　要：目的:克隆人肠道病毒71型(EV71)及柯萨奇病毒A组16型(CA16)的VP1蛋白编码基因,构建相应真核表达质粒在体外进行重组表达,检测其细胞内定位,为EV71及CA16的疫苗研究提供基础。方法:通过PCR扩增分别获取EV71及CA16的VP1编码序列,插入pcFlag载体,分别构建EV71及CA16 VP1与FLAG标签融合的真核表达质粒;瞬时转染人胚肾293T细胞及横纹肌肉瘤RD细胞,Western blot法检测融合蛋白的表达,免疫荧光检测融合蛋白的细胞内定位情况。结果:测序表明两种来源VP1蛋白的重组表达质粒构建正确;分别转染两种不同的细胞后,均可通过Western blot检测到相应蛋白表达;免疫荧光检测显示两种毒株的VP1均表达在细胞质中。结论:外源融合表达EV71或CA16 VP1蛋白的真核表达质粒构建成功;所构建质粒分别转染细胞后,均可在细胞质内检测到VP1融合蛋白的表达。OBJECTIVE: To clone VP1 genes from enterovirus 71 (EV71) and coxsackievirus A16 (CA16) and to express the genes in eukaryotic cells. METHODS:VP1 coding sequences were obtained by PCR, and then inserted into pcFlag vector to construct FLAG-fused eukaryotic expression plasmids. The recombinant plasmids were used to transfect 293T cells and RD cells,and Western blotting was performed to detect the recombinant proteins. RESULTS: The plasmids containing VP1 were confirmed by sequencing. Recombinant proteins were detected in transfected cells using Western blotting. CONCLUSION: Exogenous expression of plasmids containing VP1 coding sequences of EV71 and CA16 were constructed successfully,and the recombinant proteins was detected in transfected cells.

关 键 词：肠道病毒 EV71 柯萨奇病毒 VP1 真核表达 

分 类 号：Q786[生物学—分子生物学]