Construction And In Vitro ANTI-HBV Research Of Eukaryotic Expression Vector PCMV-HA-Tollip

There is a high incidence of hepatitis B virus (HBV) infection in China,and persistent infections with HBV lead to cirrhosis and liver cancer, whichis severely harmful to human health. Therefore, the prevention of HBVinfection is becoming a hot topic of infectious disease research in China.Although hepatitis B vaccine has a significantly protective effect on thosewho has not been infected by HBV, there are still many difficulties in thetreatment of hepatitis B, especially for chronic infection. At present, thenucleotide drugs and interferon are widely used in anti-HBV infection, butthe lack of effective long-term response effects and individual resistancefactor, restricted its use. It is urgent to found new therapeutic targets anddrugs. Toll like receptors (TLRs), as an important pathogen patternrecognition receptors, are able to identify HBV and other viruses.TLRs/IL-1R signal transduction pathway plays an important role ininflammation and innate immune. Key molecules in this signaling pathwaymay become new targets for anti-HBV infection. Tollip (Toll-interactingprotein) is a critical adapter protein in this pathway, and it is worth to explore the effect of Tollip in HBV infection.In this research, we constructed Tollip eukaryotic expressed vectorpCMV-HA-Tollip with HA-tagged protein, then transfected into humanhepatoma cell line HepG2.2.15successfully. We observed the effects ofpCMV-HA-Tollip on HBV antigens secretion and analyzed the expressionof important molecules in TLRs/IL-1R signaling pathway, to clarify themechanism of Tollip in chronic hepatitis and provide new therapeutictargets for the treatment of hepatitis B.Objective：To construst the recombinant expression plasmidcontaining HA label protein—pCMV-HA-Tollip and transfect it intoHepG2.2.15cells; To study the effects of pCMV-HA-Tollip on hepatitis Bsurface antigen and hepatitis B e antigen expression in vitro and to explorethe molecular mechanism．Methods：1．pCMV-HA-Tollip was constructed and identified itwith double restriction enzyme digestion and DNAsequencing．2．pCMV-HA-Tollip was transfected into HepG2.2.15cellsby LipofectamineTM2000．3．ELISA was used to detected the changes ofHBsAg and HBeAg in HepG2.2.15cells．4．Protein expression of AKT,NF-κB and p-AKT were determined by Western Blot．Results：1.pCMV-HA-Tollip was successfullyconstructed．2.pCMV-HA-Tollip was transfected into HepG2.2.15cellssuccessfully.48hours later, compared with the control group of6μg empty plasmid，the levels of HBsAg and HBeAg in group of3μg emptyplasmid and3μg pCMV-HA-Tollip transfected and group of6μgpCMV-HA-Tollip transfected were reduced by24%and41%(p＜0.01) and13%and31%(p＜0.01)，respectively．The levels of NF-κB and p-AKTwere also reduced(p＜0.01), but there was no apparent change of AKTexpression.Conclusion：1.The recombinant expression plasmid containing HAlabel protein—pCMV-HA-Tollip was constructed successfully.2.Tollipcould effectively inhibit the secretion of HBsAg and HBeAg of HepG2.2.15cells by reducing the NF-κB and p-AKT expression in vitro．...