Construction of pEGFP-C1-JEV eukaryotic expression vector of E gene from JEV and expression fusion protein molecule in BHK-21 cells
10.3760/cma.j.issn.0254-5101.2012.07.007
- VernacularTitle: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
- Author:
Ruofei FENG
;
Xiaoyuan ZHANG
;
Guihong LIN
;
Mingsheng LI
;
Zilin QIAO
;
Jingjing FAN
;
Xiangrong LI
;
Haixia ZHANG
;
Deying FAN
;
Zhongren MA
- Publication Type:Journal Article
- Keywords:
Japanese encephalitis virus;
E gene segment;
Green fluorescent protein;
Eukaryotic expression
- From:
Chinese Journal of Microbiology and Immunology
2012;32(7):600-605
- CountryChina
- Language:Chinese
-
Abstract:
Objective To research the molecular biology characteristics and transient expression in BHK-21 cells of E geue segment from Japanese encephalitis virus(JEV) and construct an eukaryotic expression vector pEGFP-C1-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 transcription of aim gene,and to check up localization and antigenicity of expressed E protein by Immunohistochemistry and Western blot.Results It showed that the recombinant plasmid pEGFP-C1-JEV was successfully constructed and transfected 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 protein 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 antigenicity.This research as a base foundation for E protein gene of JEV eukaryotic expression and function in vitro and applied research.
