TI:Construction of eukaryotic expression vector expressing hepatitis C virus NS5B and EGFP fusion protein and establishment of stable transfected HepG2 cell line
AU:LEI Yingfeng , XUE Xiaoping , YIN Wen , LB Xin ,YANG Jing
AD:Department of Microbiology , The Fourth Military Medical University , Xi’an 710032 ,China Corresponding author : XUE Xiao2ping , E2mail : xue- xiaoping @yahoo. com
【Abstract】　Objective 　To construct a eukaryotic expression vector for expressing hepatitis C virus (HCV) recombinant NS5B2EGFP fusion protein and obtain a stable transfected HepG2 cell line1 Methods 　The coding region of NS5B gene of HCV was amplified by PCR and was digested by Xho ⅠPKpn Ⅰ1 This fragment was inserted into pEGFPN3 with T4 ligase and transformed E1coli TG11 The positive recombinant plasmid was selected , then the recombinant plasmid was transfected into HepG2 cell by Lipofect AMINE 20001 Cells containing stable transformants were selected by the ability of resistance to G418 and isolated with a limited dilution1 The stable transfected cell line expressing high level NS5B2EGFP fusion protein was obtained1 Results 　The eukaryotic expression vector named pEGFPN32ns5b was successfully constructed and the stable transfected HepG2 cell line expressing NS5B2EGFP fusion protein was obtained1 Conclusion 　The stable transfected HepG2 cell line could express NS5B2EGFP fusion protein , could be used for anti2HCV infection with ns5b gene as the target1
【Key words】　Hepacivirus ; 　Viral fusion proteins ; 　Transfection ; 　Cell line