Construction of recombinant lentiviral vector expressing HCV core-Ant / 西安交通大学学报(医学版)

ABSTRACT

Objective To construct a recombinant lentiviral vector for HCV core-Ant and then study its effect on the transdifferentiation of hepatic stem cells. Methods The HCV core-Ant was obtained by PCR of two primers template with each other and T-vector cloning method,and then subcloned to pLenti6/V5-D-TOPO. The restriction endonuclease and T4 DNA ligase were used to construct the vector. pLenti6/V5-D-HCV core-Ant and the ViraPowerTM PackagingMix (containing three packaging plasmids pLP1,pLP2 and pLP/VSVG) were cotransfected into 293ET cells to produce replication in competent lentivirus after transfection. The viral supernatant on 293T cells was collected. The expression of the lentiviral vector containing HCV core-Ant in Hela cells was measured by immunohistochemistry. Then we constructed the lentiviral vector containing green flurosecent protein by the similar method,and the titers were determined. Results HCV core-Ant was identified and analyzed by restriction enzyme digestion and sequencing,respectively. The expression of the recombinant lentiviral vector plasmid containing HCV core-Ant in Hela cells was confirmed by immunohistochemistry. The 3T3 cells transfected the lentiviral vector containing green flurosecent protein were found to show strong expression of GFP,which confirmed that the four-plasmid system of the lentiviral vector was successfully constructed. Conclusion The recombinant lentiviral vector expressing HCV core-Ant was successfully constructed by molecular cloning and recombination techniques in vitro,which will be beneficial to guiding further study on gene therapy of cancer.