Producing the Recombinant Structural Protein of Chicken Anemia Virus and Chicken Interleukin-12 as Vaccine Candidates by Baculovirus Expression System

• Main Authors: Ta-Yuan Tseng, 曾達元
• Other Authors: 歐繕嘉
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/g32ef6

碩士 === 國立中興大學 === 微生物暨公共衛生學研究所 === 106 === Chicken anemia virus（CAV）is one of the important pathogens affecting poultry industry globally. The virus causes mortality, production losses, immunosuppression, sensitive to infections and vaccination failures in infected chickens. Currently, only live attenuated chicken infectious anemia（CIA）vaccines are available for administering in breeders older than 8 weeks that induce maternal antibodies to protect their progeny till 3 weeks. However, the vaccine viruses could induce vertical and horizontal infection to other chickens. These disadvantages of the traditional vaccines induce the idea to develop novel CIA vaccines. According to previous studies, VP1 is the only structural protein forming the viral capsid, and the correct folding of VP1 requires expression of VP1 and VP2 in the same cell. Interleukin-12（IL-12）is a proinflammatory cytokine, which is responsible for enhancing cytotoxicity of NK cells and T cells. Moreover, IL-12 facilitates the generation of related TH1 cytokines, and promotes the production of immunoglobulins by stimulating helper T cells. Therefore, IL-12 can be serve as a biological adjuvant. In this study, the baculovirus expression system was used to produce the structural protein of CAV and chicken IL-12（chIL-12）for developing a subunit vaccine candidate. The VP1 and VP2 of CAV and the chIL-12 gene codons were optimized for expressing in the insect cells, and the recombinant proteins were expressed by the baculovirus expression system. Western blot and indirect immunofluorescence assays were used to verify the expression of recombinant proteins. The results showed that VP1 and chIL-12 can be expressed in eukaryotic cells. We found that VP1 predominantly aggregated in the nucleus, and the amount of expression level needs to be further optimized. Biological adjuvant property of chIL-12 has been tested by inducing high expression level of IFN-γ in the recombinant chIL-12 stimulated splenocytes. Finally, different concentrations of recombinant VP1 and chIL-12 has been combined together to assess immune responses in chickens, and the results showed that VP1 combined with chIL-12 achieved the highest titers of CAV specific antibody than those of other groups. Taken together, the recombinant VP1 could be a candidate of CAV subunit vaccine, and co-immunization of chIL-12 can induce a strong immune response in vaccinated chickens.