ABSTRACT
<p><b>OBJECTIVE</b>To construct a eukaryotic expression vector for expressing hepatitis C virus (HCV) recombinant NS5B-EGFP fusion protein and obtain a stable transfected HepG2 cell line.</p><p><b>METHODS</b>The coding region of NS5B gene of HCV was amplified by PCR and was digested by Xho I/Kpn I. This fragment was inserted into pEGFPN3 with T4 ligase and transformed E. coli TG1. The positive recombinant plasmid was selected, then the recombinant plasmid was transfected into HepG2 cell by Lipofectin AMINE 2000. Cells containing stable transformants were selected by the ability of resistance to G418 and isolated with a limited dilution. The stable transfected cell line expressing high level NS5B-EGFP fusion protein was obtained.</p><p><b>RESULTS</b>The eukaryotic expression vector named pEGFPN3-ns5b was successfully constructed and the stable transfected HepG2 cell line expressing NS5B-EGFP fusion protein was obtained.</p><p><b>CONCLUSION</b>The stable transfected HepG2 cell line could express NS5B-EGFP fusion protein, could be used for anti-HCV infection with ns5b gene as the target.</p>
Subject(s)
Humans , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Genetic Vectors , Genetics , Green Fluorescent Proteins , Genetics , Metabolism , Microscopy, Fluorescence , Recombinant Fusion Proteins , Genetics , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Viral Nonstructural Proteins , Genetics , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To identify and characterize the epitope associated with the virus attachment protein (VAP) of hantaan virus.</p><p><b>METHODS</b>The monoclonal antibody 3G1 was used as the ligand to biospan from a phage-displayed 12-amino acid peptide library, then the positive phage clones were chosen and sequenced. The amino acid sequences of them were compared with that of hantaan virus G2 in homology. The characteristics of positive phage were studied by IFA and ELISA. A decapeptide combining to cell membrane was observed under laser scanning confocal microscope (LSCM).</p><p><b>RESULTS</b>The conservative motif PX(1-2) HX(0-2) H displaying on positive clones shared homologous amino acid sequence with G2 96YPWHTAKCHY105.</p><p><b>CONCLUSION</b>G2 96YPWHTAKCHY105 might play some roles in virus binding to host cell, and might be a possible key epitope of hantaan virus VAP.</p>