Construction and investigation of a recombinant eukaryotic expression vector for expressing the ORF3 protein of hepatitis E virus in BHK-21 fibroblasts / 中华肝脏病杂志
Chinese Journal of Hepatology
;
(12): 499-503, 2014.
Article
in Chinese
| WPRIM
| ID: wpr-314014
ABSTRACT
<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>The 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.</p><p><b>CONCLUSION</b>The 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.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Plasmids
/
Viral Proteins
/
Recombinant Proteins
/
Transfection
/
Cell Line
/
Open Reading Frames
/
Hepatitis E virus
/
Cytoplasm
/
Fibroblasts
/
Genetic Vectors
Limits:
Animals
Language:
Chinese
Journal:
Chinese Journal of Hepatology
Year:
2014
Type:
Article
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