title: [Prokaryotic expression of Hepatitis C Virus (HCV) NS3 protein and preparation of polyclonal antibody].
abstract: To increase detection sensitivity and specificity on hepatitis C virus (HCV) is vital for prevention and controlling of the disease. To establish a more reliable detection method for HCV diagnosis, the full gene fragment of ns3 (non-structural protein of HCV) from recombinant plasmid of J6/JFH1 2a was amplified and then connected into the pET-28a prokaryotic expression vector, and the latter was subsequently transformed into Escherichia coli BL21 (DE3) to have the target protein expression. As a result, a protein with a molecular weight of 72 kDa was obtained and visualized in 10% SDS-PAGE. The purified NS3 protein was used as immunogen to inoculate BALB/c mice and the sera was collected after the fourth immunization. The antibody titer of serum is determined to be about 1:256000 with ELISA. Western blotting and indirect immunofluorescence analysis showed that the mouse polyclonal antibody could react specifically with the native NS3 protein in Huh 7.5.1 cells infected with HCV. These findings may provide basis for further preparation of monoclonal antibodies against NS3 and the development of related detection kit.
authors.forename: Tao
authors.initials: T
authors.lastname: Sun
authors.forename: Guangwen
authors.initials: G
authors.lastname: Yang
authors.forename: Jinyang
authors.initials: J
authors.lastname: Zhang
authors.forename: Xueshan
authors.initials: X
authors.lastname: Xia
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D016174:Hepacivirus; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis* / Q000276:immunology*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000914:Antibodies, Viral; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases