title: [Construction and investigation of a recombinant eukaryotic expression vector for expressing the ORF3 protein of hepatitis E virus in BHK-21 fibroblasts].
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Infectious Diseases, the First Affiliated Hospital of Lanzhou University, Lanzhou 730000, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Chen
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Yang
authors.forename: Hong
authors.initials: H
authors.lastname: Yuan
authors.forename: Longdong
authors.initials: L
authors.lastname: Zhu
authors.forename: Wei
authors.initials: W
authors.lastname: Yue
mesh_terms: D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003593:Cytoplasm; D005347:Fibroblasts / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics*; D016752:Hepatitis E virus / Q000378:metabolism*; D008164:Luminescent Proteins; D016366:Open Reading Frames; D010957:Plasmids; D011994:Recombinant Proteins; D014162:Transfection; D014764:Viral Proteins / Q000378:metabolism*; D000097573:Red Fluorescent Protein
chemical_list: D008164:Luminescent Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; D000097573:Red Fluorescent Protein; C400973:ORF3 protein, Hepatitis E virus