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>
Subject(s)
Animals , Cricetinae , Cell Line , Cytoplasm , Fibroblasts , Metabolism , Genetic Vectors , Genetics , Hepatitis E virus , Metabolism , Luminescent Proteins , Open Reading Frames , Plasmids , Recombinant Proteins , Transfection , Viral Proteins , MetabolismABSTRACT
0.05). Serum glutamate pyruvate transaminase activity significantly decreased in the injured liver BMSCs group (P