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.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
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）
关键词
乙型脑炎病毒
E蛋白基因
绿色荧光蛋白
真核表达
Japanese encephahtls wrus
E gene segment
Green fluorescent protein
Eukaryotmexpression
