| Hepatitis B virus(HBV) is a serious worldwide public health problem. The prevalence rate is about 10% and there are approximately thirty million people suffered from chronic hepatitis in China. In addition to causing acute hepatitis or chronic hepatitis, HBV can eventually cause cirrhosis and hepatocellular carcinoma in part of people infected by it. 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. HBsAg encoded by the s gene is the major component of Hepatitis B vaccine. It is also an important diagnostic evidence of HBV infection. Hepatitis B immunogloblin could be used to protect the liver from reinfection after liver transplantation, but it is 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 is most commonly to find the mutation at aa!45 where Glycocol substituted by arginine(G145R). This mutant could result in the failure of HB vaccination and failing to protect the liver from reinfection after liver transplantion, as well as vertical transmission and so on. It brings about many new problems to prevention and cure of HB. It necessary to develop HB vaccine against this mutant and to do some further reseach on the change of biological character caused by this point mutation. In addition, hepatitis B virus belongs to hepadnaviridae and has narrow host. In natural condition, HBV could only infect human being and higher primate, but could not infect mammal such as mouse and other rodent which is commonly used in scientific reseach. Cultural cells could only used in vitro. Therefore, it prevents the further study on mutation of HBV in some degree. The generation of trans genie mice harbouring the G145R mutant could provide a good animal model for the overall study on change of antigenicity and immunogenicity as well as pathogenicity of HBV caused by this point mutation. It could also provide good animal model for drug screen and therapy involved in this mutant.The recombinant eukaryotic expression vector(PR) containing the mutant s gene at the site of nt 587 (G-A) was constructed. Restriction endonucleases was used to confirm the vector and the sequence of s gene in PR was determinated. Then PR was transfected into human hepatocellular carcinoma cells (Hep G2) via electrotransformation. And then it was used to immunize C57BL/6 normal mice by way of intramuscular injection. It was found that HBsAg in supernatant of transfected cells detected by EIA was positive at the third day after tranfection and the OD value increased as time goes on. But the OD value of the mutant HBsAg was appearantly lower than the one of the wild-type. Both mutant-type and wild-type HBsAg had reaction to anti-HBs in histocytochemical staining. There was no significant differentiation in two groups. PR can stimulate an immune response of anti-HBs and anti-HBs2 in normal mice. The occure of protective antibodies of anti-HBs in PS group was one to two weeks earlier than that in PR group.The occure of protective antibodies of anti-HBs2 was one to two weeks earlier than that of anti-HBs in the same group.The average litres of anti-HBs in PS group was higher than that in PR group; but the average titres of anti-HBs2 has no significant differentiation in the twogroups. HBsAg specific lymphocyte activity of mice 12 weeks after immunization was measured by 51Chromium release assay. The strong HBsAg specific cytoticity mediated by lymphocytes was detected. All the results showed that the mutant HBsAg expressed by PR has strong antigenicity, and has reacting with anti-HBs antibody in vitro. But the affinity is obviously lower than that of the wild type. The mutation of s gene reduces the antigenicity and immunogenicity of HBsAg PR could still stimulate an strong humoral immune respone in mice, but.In or...
|
PDF Full Text Request