Expression, Purification And Immunocompetence Research Of A Mutational HBsAg In Baculovirus Expression System

Hepatitis B virus (HBV) is a serious worldwide public health problem. In China, HBV carriers account for 10% of total population and there are about 30 million people suffered from chronic hepatitis. In addition to causing acute hepatitis or chronic hepatitis, HBV often eventually lead to cirrhosis and hepatocellular carcinoma in some patient. Genetic mutation in viruses are usually associated with escape of host immune responses, developing drug resistance, modification of virulence and patterns of epidemiology of diseases. Therefore, study on viral mutations are of great importance in prophylaxis and therapy of HBV. S gene encoded HBsAg is the major component of hepatitis B vaccine, and it is also the most diagnostic evidence of HBV infection. Hepatitis B immunoglobulin could be used to protect the patient from reinfection after liver transplantation, but this effect rely on the interaction of HBsAg and anti-HBs. Therefore, the change of antigenicity of HBsAg has a direct impact on the diagnosis and therapy of HBV. It's well known that the most common mutation at aal45 where glycine is substituted by arginine(G145R) will result in the failure of HBV vaccination, transplantation protection, and vertical transmission protection.In this study, we isolate a G145R mutant (X119) from serum of a blood donor, amplify its S gene with PCR method and clone into a recombinant baculovirus vector for mass production of this variant HBsAg (119s). The protein is purified by HPLC and CsCl equilibriumcentrifugation. Transmission electron microscopy investigation indicates that the purified 119s could form virus like particles autonomously. In order to investigate the immunogenicity of the recombinant antigen, we use purified 119s to immune mice and detect intense immune response and specific antibody generation compared to control group. Above all, the variant HBsAg produced by recombinant baculovirus vector system may be used as a new component of HBV vaccine to provide protection against G145R mutant strain. Structure analyses indicate that the variant HBsAg exhibits significantly different conformation compared with wildtype HBsAg and this may contribute to the insensitive of current diagnostic agent in detecting variant strain of HBV. New diagnostic agent contained recombinant variant antigen may help promote the detection rate of variant HBV.