| Hepatitis B virus (HBV) is a major public health problem with more than 300 million people infected worldwide, and a much greater proportion (around 5-10% of adults and 80-90% of young infected children) fail to clear the virus and go on to become chronic hepatitis or carrier. Chronic hepatitis or carrier is a risk of developing chronic cirrhosis and hepatocellular carcinoma and represent the primary reservoir of infection. Our country is a high-risk prevalent region of HBV, and the total amount of the chronic HBV carrier exceeds 100 million (about 10% of population). Prevention and treatment of the chronic hepatitis B is still a worldwide puzzle. The two main therapeutic options for it, interferon- α (IFN- α ) and lamivudine, are unsatisfactory. Although they can inhibit replication of viral DNA and exhibit antiviral activity, the efficacy of treatment is only 20%-40%, with its serious drawback and prolonged therapy, it may cause the emergence of a mutant and drug-resistant strain of HBV. However, the vaccination is thought to be the only medical measure likely to have a significant impact on the problem of HBV infection.The currently available vaccines offer the adequate protection against HBV. Since the World Health Organization (WHO) recommended that Hepatitis B vaccine should be integrated into national infant and adolescent immunization programmes in all countries by 1997, over 110 countries haveadopted national or regional policies of universal infant or adolescent immunization so far, and has the rate of HBV infection reduced remarkably. Unfortunately the current vaccine only comprised S protein requires multiple dosing and a 6-month commitment, and its incidence rate of non/sub-response reach at 10%-15%, especially it can't induce sufficient cellular responses to inhibit viral reproduction or clear the virus. So newer vaccines that are still in development may offer simpler vaccination schedules and induce immune response in patients who have failed in the standard treatment regimen or have therapeutic effect on the chronic hepatitis B.Chronic HBV infection is associated with T cell hyporesponsive- ness or tolerance and has been proposed that elimination of virus is due to CTL-mediated lyses of infected hepatocytes and/or antiviral effects of CTL-derived cytokines, such as IFN- γ and TNF- α . DNA vaccine is a potent approach to induce vigorous cellular and humoral responses, and is used widely in the study of the therapeutic vaccine. The minigene of the multiple CTL epitopes and PADRE can break the T cell tolerance and induces vigorous, multiclonal and multispecific cellular responses. The multiepitope minigene encoding preS2, PADRE, CpG and five dominant HLA-A2, A3 or B7 restricted CTL epitopes derived from the envelope, core protein and polymerase of HBV was designed and synthesized.HBc protein holds a unique position among the VLP carrier because of its high-level expression and efficient particle formation in homologous and heterologous expression systems. In addition, the HBc carrier moiety could provide T cell help to inserted sequences, and mediates the T-cell-independent character of the humoral response to inserted epitopes. In general, it is widely accepted now that the HBc carrier is capable of ensuring a high level of B cell and T cell immunogenicity to foreign epitopes. In this study, the synthetic multiepitope gene of HBV was inserted into and substituted the gene of HBc spinal region. The chimeric HBc gene then cloned into pcDNAS.l, forming the HBcMep recombinant plasmid. The-recombinant was verified by the restriction digestion and sequencing. Sequence analysis showed that the size of gene was 885bp without any introns and coding 292 amino acids. By comparing with the gene of HBV adr isotype, there are only three point mutations occurred in the cloned HBc gene, and the deduced amino acid sequences of HBc and multiepitope gene are completely same. The results demonstrated that the chimeric HBc gene carrying HBV multiepitopes is cloned correctly.It is t...
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