The Study Of IFN-a Induced By PBMCs Stimulated By HBcAg In Healthy Subjects And HBV Carriers

Objective:To construct recombinant adenoviral vector carrying HBcAg gene by homologous recombination in bacteria and to transfect human peripheral blood mononuclear cells from healthy subjects and HBV carriers, analyze the value of IFN-αin culture supernatants by ELISA and compare to the results of CpG ODN 2006 (ligand of TLR9) in purpose of investigating the mechanism of hepatitis B virus infection recognition in immune system.Methods:HBV C genes were amplified by PCR and were cloned to adenoviral shuttle plasmid pAdTrack-CMV. Then the resultant pAdTrack-CMV-HBc was cotransfected into BJ5183 bacteria with the plasmid pAdeasy-1. The adenoviral plasmid carrying HBV C gene (pAd-HBc) was generated with homologous recombination in bacteria and the adenoviruses were produced in 293 T cells. The recombinant adenoviruses Ad- HBc were obtained. With the method of density gradient centrifugation,isolating peripheral blood mononuclear cells from healthy subjects(HBsAg negative) and HBV carriers were divided into four groups. Three groups was stimulated respectively by Ad-HBc+CpG ODN 2006,Ad-HBc,CpG ODN 2006 and the fourth was control group. After 48 hours, the value of IFN-αin culture supernatants was detected by ELISA.Results:In healthy subjects, compared with control, the concentration of IFN-αin Ad-HBc+CpG ODN 2006 group,Ad-HBc group and CpG ODN 2006 group were all elevated(P<0.05),Ad-HBc+CpG ODN 2006 group increased significantly,Ad-HBc group followed,CpG ODN 2006 group the lowest. In HBV carriers, compared with control, Ad-HBc + CpG ODN 2006 group increased slightly ,CpG ODN 2006 group followed, Ad-HBc group the lowest (P<0.05).The IFN-αconcentration of Ad-HBc in HBV carriers is significantly lower than healthy subjects(P<0.05).Conclusions:Replication- deficient recombinant adenoviral vector carrying HBcAg gene infected human peripheral blood mononuclear cells from healthy subjects, increased ability of pDC can be induced to produce IFN-α.The stimulated result was more effective than the ligand CpG ODN 2006 of TLR9. In HBV carriers, the adenovirus infection of peripheral blood mononuclear cells were disable to produce IFN-αmore than CpG ODN 2006,the result may involve in dysfunction of pDCs caused by persistent infection of HBV.