The Study Of Relationship Between Impairing Natural Killer Cell Function By Hepatitis B Virus X Protein And The Establishment Of A Chronic Hepatitis B Virus Infection

Objective To investigate the influence of the expression of HBV X protein onthe function of NK cell, and hence shed new light on the mechanism of the establishment of chronic hepatits B.Methods The recombinant eukaryotic expression plasmid pcDNA3.1 (+)-HBX was confirmed by double restrictive enzyme digestion and DNA sequencing analysis. Then the recombinant plasmid was transfected into NK-92 cells with Hpofectamine encapsuled. The transfected NK-92 cells containing expressive HBV X gene was confirmed by RT-PCR and Western blotting analysis. Western blotting was also applied for the determination of NKG2D expression. Enzyme linked immunosobent assay (ELISA) was employed to determine the IFN-y level 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 colorimetry analysis, with HepG2, the hepatoblastoma cell line as the target cell.Results The sequence of pcDNA3.1(+)-HBX target gene, as identified by double restrictive enzyme digestion and sequencing, was identical to the HBV X gene sequence published in GeneBank with no mutation found. RT-PCR assay showed that a 192bp cDNA fragment, which represents HBx, was amplified from NK-92 cells transfected with pcDNA3.1(+)-HBX, while no fragment was amplified from the NK-92 cells transfected with blank plasmid and untransfected NK-92 cells. Western blotting assay showed that a 17KDa protein was separated from NK-92 cells transfected with pcDNA3.1(+)-HBX, which can specifically bind to the HBX monoclonal antibody, and no such protein was separated from the two control NK-92 cells above. The NKG2D Western blotting assay showed a dramatic decrease of...