Construction And Identification Of A Recombinant Lentiviral Vector Expressing Hepatitis B Virus X Protein

Objective To investigate the transfection efficiency of the recombinant HBVX-containing lentiviral vector in the HL7702 cell line from normal tissue .Methods The lentiviral vector was reconstructed by insert the lentiviral vectors with the HBVX gene fragment, which was isolated from pcDNA3-X vector. It was analysised with PCR restriction digestion and bidirectional sequencing.The recombination lentiviral particles were produced by the packaging 293T cell by using the jetPEI method, the culture supernatant was harvested and the titration of them were calculated by the limiting dilution.The HL7702 cells were transduced by the lentiviral particles,after 96h the GFP expression was observed under the fluorescence microscope. The expression level of HBV X in cells were examed by the Real-time PCR and Western blot.Results The recombinant lentiviral vector pRRL-X had been successfully constructed, the titration of lentivirus particles was 4X107IU/m1. The HL7702 cells could be transduced by the recombinant lentiviral particles (4X10~7IU/m1). After 96h co-culture, ,various intensities of florescence can be seen in the cell. The expression of HBVX gene in recombinant HL7702 was confirmed in the way of Real-time PCR and Western blot.Conclusion Lentiviral vectors expressing HBVX gene were successfully constructed and provide a tools for research in HBVX gene on the effects of liver cells.