the stuy of relationship between impairing natural killer cell function by hepatitis b virus x protein and the establishment of a chronic hepatitis b virus infection.pdf

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 Abstract Objective To investigate the influence of the expression of HBV X protein on the 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 lipofectamine 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-γ 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