Screening And Identification Of Candidate Epitopes For Epitope Vaccine Of Avian Leukosis Virus Subgroup J

Avain leukosis virus subgroup J(ALV-J)is an infectious tumor virus, which is a member of retrovirus family, mainly causes neoplastic proliferation of certain hematopoietic cell. In field, it mainly causes growth retardation, immunosuppression and mutiple tissue tumor in various tissues. In 1999, ALV-J was first isolated the virus from broriler breeder in China. After that, its host range was expanded, from broiler breeder to laying hens, and then local chicken. The oncogenicity was significantly enhanced and caused varieties of tumors.The incidence age was more early, appearing lymphosarcomars, hemangiomas,fibrosarcomas, and erythroblastomas, causing serious economic losses in poultry industry.Considering the characteristics of hypervariation and the immune evasion mechanisms of the virus, the traditional attenuated vaccine and inactibated vaccine do not provide effective protection. Developed countries performs constantly eliminating the ALV-J positive chicken to prevent the disease. Due to the relative low breeding level and the slow development of poultry industry, the disease caused by ALV-J cann’t be completely controlled by eliminating the positive chicken. Currently, ALV-J is prevalent in China, it is in urgent need to develop new effective vaccines.Epitope vaccine is a novel subunit vaccine that developed quickly. It has many advantages and can overcome the virulence recovery and shedding caused by the traditional vaccines. Epitope peptides are short but more immunogenic, can overcome the genetic restriction caused by the major histocompatibility complex. Multi-epitope vaccine contains a broad spectrum of epitope information, which can provide a more complete and more targeted immune protection to overcome the inoculation failure caused by vatiation, and provent the virus spreading. Screening and identification of more epitopes, not only help to understand the virus antigen structure, also complete a hard job for the research ofepitope-based vaccine.In this study, more about virus structure and antigenic information were refered,epitopes of ALV-J envelope were predicted by analysis. Epitopes sequences were screened,and can be on behalf of the Chinese pandemic strains in recent years. Different epitope genes were connected by codons which encode glycine and serine, and synthesized by Sangon biotechnology company. We choose different prokaryotic expression vectors for expressing recombinant protein, and the highest purity is the opimized choice. After the recombinant protein was confirmed highly expressed and well purificated by SDS-PAGE,immunogenicity was analylized by Western Blot, finally the animal immunization experiment was performed.The recombinant protein was confirmed immunogenic by animal experiment, and can stimulate the chicken body to produce effective antibodies. What’s more, the antibody can resist for 14 weeks. Virus neutralizing experiment was performed by using the prepared effictive serum. The results showed that, the antibody has an obvious effect on virus neutralization, and neutralizing rate can reach up to 40%-50%. For this mutated virus, it is necessary to further pinpoint epitope in hypervariable region, and improve the epitope mapping, enhancing the immune protecting effect. Eight different segments of the gp85 hypervariable region were expressed in prokaryotic expression, then their immunogenicity was analylized with specific monoclonal antibody 2D5 by indirect ELISA. Epitope region was identified first, epitope mapping was further performed with syntheticing peptides.Finally, we can precise the epitope key amino acid sequence of the hypervariable region is LRDFIAKWKSDDLLIRPYVNQS.Bioinformatic analysis of the epitope amino acid sequence showed that there were mainly three sites of variation in the epitope. By further indirect ELISA analysis with different sequence peptides, the key amino acid was confirmed, which lied in the sixth site of the epitope, presenting A to E or T. We analyzed the immunogenicity of variaty epitope peptides by clinical ALV-J serum, the result showed that different epitope peptides onlyreact with positive serum, we can conclude the identified epitope has reactogenicity and can speculate the chicken infected ALV-J produce multiple antibodies.In this study, the idedtified epitopes enriched ALV-J epitope spectrum, and will provide a powerful tool to develop effective multi-epitope vaccine and prevent the diseases.