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

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. Key words: Japanese encephalitis virus; E gene segment; Green fluorescent protein; Eukaryotic expression