乙型脑炎病毒E蛋白基因真核表达载体pEGFP-C1-JEV的构建及在BHK-21细胞中表达  

Construction of pEGFP-C1-JEV eukaryotic expression vector of E gene from JEV and expression fu- sion protein molecule in BHK-21 cells

作　　者：冯若飞[1] 张晓媛[2] 林贵鸿[1] 乔自林[1] 凡静静[3] 李向茸[3] 张海霞[3] 樊得英[1] 马忠仁[1] 

机构地区：[1]西北民族大学生物工程与技术国家民委重点实验室,兰州730030 [2]河北北方学院基础医学院 [3]西北民族大学生命科学与工程学院

出　　处：《中华微生物学和免疫学杂志》2012年第7期600-605,共6页Chinese Journal of Microbiology and Immunology

基　　金：甘肃省中青年科技基金计划项目（0806RP/A008）

摘　　要：目的构建乙脑病毒E蛋白基因片段的真核表达载体pEGFP-C1-JEV，旨在研究E蛋白基因在BHK-21细胞中的瞬时表达情况，为进一步深入研究提供基础资料。方法通过liT—PCR扩增目的基因，酶切并重组后成功构建绿色荧光蛋白标记的pEGrP—C1-JEV重组表达载体，利用脂质体介导将重组质粒转染BHK-21细胞，观察绿色荧光标记蛋白表达情况，同时提取细胞RNA，检查目的基因转录表达情况，并利用免疫组化和Westernblot法检测目的基因表达情况、表达蛋白在细胞中的定位以及表达蛋白抗原性。结果重组质粒pEGrP—C1-JEV构建成功，转染至BHK-21细胞中，绿色荧光标记蛋白正常表达，表达率较高，RT-PCR表明目的基因在BHK-21正常转录并表达，表达蛋白主要分布在BHK-21细胞的胞质和包膜中，且能与豚鼠抗JEV抗体特异性结合。结论pEGFP-C1-JEV质粒在BHK-21细胞中正常表达，且对细胞生长和形态不产生影响，真核表达抗原具有良好的抗原性，本研究可为进一步探讨乙型脑炎E蛋白基因体外真核表达及其功能和应用的研究提供基础资料。Objective To research the molecular biology characteristics and transient expression in BHK-21 cells of E gene segment from Japanese encephalitis virns（JEV） and construct an eukaryotic ex- pression vector pEGFP-CI-JEV. Methods E gene segment of JEV was amplified by RT-PCR, construct the recombinant vector pEGFP-C1-JEV, which could express EGFP label proteins. Transfect pEGFP-C1-JEV vector into BHK-21 via LipofectAMINETM 2000, to observe expressing of EGFP label protein and tmnscfip- tion of aim gene,and to check up localization and antigenicity of expressed E protein by Immunohistochemis- try and Western blot. Results It showed that the recombinant plasmid pEGFP-C1-JEV was successfully constructed and tran_sfected to BHK-21 cells, the normal expression of green fluorescent protein expression rate was higher. RT-PCR showed that gene transcription in BHK-21 and normal expression, expression pro- tein was mainly distributed in the cytoplasm of BHK-21 cells and the envelope in, and can with guinea pig anti-JEV antibody binding. Conclusion pEGFP-C1-JEV vector in BHK-21 cells was normal expression and there were no effect on cell growth and morphology. Meanwhile, on eukaryotic antigens was good antigeniei- ty. This research as a base foundation for E protein gene of JEV eukaryotic expression and function in vitro and applied research.

关 键 词：乙型脑炎病毒 E蛋白基因 绿色荧光蛋白 真核表达 

分 类 号：R3[医药卫生—基础医学]