Construction and investigation of a recombinant eukaryotic expression vector for expressing the ORF3 protein of hepatitis E virus in BHK-21 fibroblasts.
- VernacularTitle:戊型肝炎病毒开放读码框3真核表达载体构建及其在BHK-21细胞中的表达
- Author:
Lin CHEN
1
;
Xiaojun YANG
;
Hong YUAN
;
Longdong ZHU
;
Wei YUE
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Cell Line; Cricetinae; Cytoplasm; Fibroblasts; metabolism; Genetic Vectors; genetics; Hepatitis E virus; metabolism; Luminescent Proteins; Open Reading Frames; Plasmids; Recombinant Proteins; Transfection; Viral Proteins; metabolism
- From: Chinese Journal of Hepatology 2014;22(7):499-503
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct a eukaryotic expression vector to express the hepatitis E virus protein open reading frame 3 (ORF3) and investigate the intracellular location of the expressed protein using the baby hamster kidney (BHK-21) fibroblast cell line.
METHODSThe ORF3 gene was amplified by RT-PCR, cloned into the HindIII and EcoRI sites in the multicloning site of the pDsRed-Monomer-N1mammalian expression vector that encodes a red fluorescent protein (DsRed), and confirmed by restriction enzyme digestion and sequencing. The recombinant plasmid was then transfected into BHK-21 cells via the Lipofectamine 2000 reagent; the subsequent ORF3 gene overexpression was confirmed by RT-PCR and the protein expression and location was detected by Western blotting and immunofluorescence assay.Results TThe pDsRed-Monomer-N1-ORF3 recombinant plasmid was successfully constructed. After transfection into BHK-21 ceils, the ORF3 gene was transcribed and expressed, and the ORF3 protein was mainly located in the cytoplasm, where it could react with a specific antibody.
CONCLUSIONThe ORF3-DsRed fusion protein was mainly located in the cytoplasm of BHK-21 fibroblasts, and may represent a useful tool for research on the role of this protein in HEV infection.