The Study Of Influence Of Hepatitis B Virus C Protein On The Function Of Natural Killer Cell

Objectives To investigate the influence of hepatitis B virus C protein on the function of natural killer cell, and hence shed new light on the mechanism of the establishment of chronic hepatits B.Methods Recombinant eukaryotic expression plasmid pHBI-CMV-HBC was constructed and confirmed by double restrictive enzyme digestion and DNA sequencing analysis. Then the recombinant plasmid was transfected into NK-92 cells with lipofectamine encapsuled. The transfected NK-92 cells containing expressive HBV c protein was confirmed by Western blotting analysis. Enzyme linked immunosobent assay(ELISA) was employed to determine the IFN-γlevel secreted by NK-92 cell in the cell culture supernatant. Then the effect of eukaryotic expression plasmid containing target gene on NK-cell cytotoxic activity was examined by MTT assay, with HepG2, the hepatoblastoma cell line as the target cell.Results The sequence of HBV C DNA,as identified by double restrictive enzyme digestion and sequencing, was identical to the HBV C gene sequence published in GeneBank with no mutation found. Western blotting assay showed that a 21KDa protein was separated from NK-92 cells transfected with pHBI-CMV-HBC, which can specifically bind to the HBC monoclonal antibody, and no such protein was separated from the two control NK-92 cells above. MTT assay showed that the killing rates in NK-92 cells transfected with recombinant plasmid was significantly higher than that of NK-92 cells transfected with blank plasmid (P<0.01) and untransfected NK-92 cells(P<0.01). ELISA showed an obvious increase in IFN-γlevel secreted by NK-92 cell transfected with pHBI-CMV-HBC, compared with NK-92 cells transfected with blank plasmid(P<0.01)and untransfected NK-92 cells(P<0.01).Conclussion1. pHBI-CMV-HBC can be transfected into NK cell successfully with Lipofectamin and the target gene can be expressed in it.2. The transient expression of HBV C gene can increase the cytotoxity of NK-92 cell to HepG2 cell.3. The transient expression of HBV C gene can increase IFN-γsecretion of NK-92 cell.