Construction and investigation of a recombinant eukaryotic expression vector for expressing the ORF3 protein of hepatitis E virus in BHK-21 fibroblasts].

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. 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. 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.

Citation

Lin Chen, Xiaojun Yang, Hong Yuan, Longdong Zhu, Wei Yue. Construction and investigation of a recombinant eukaryotic expression vector for expressing the ORF3 protein of hepatitis E virus in BHK-21 fibroblasts]. Zhonghua gan zang bing za zhi = Zhonghua ganzangbing zazhi = Chinese journal of hepatology. 2014 Jul;22(7):499-503

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PMID: 25203800

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