The Immunoregulation Of Gallus Akirin2 And GM-CSF Gene On IBDV VP2 DNA Vaccine

Akirins encodes a nuclear protein that is required downstream in the TLR pathway at the level of NF-?B, is a potential biological adjuvant that plays a critical role in the immune and inflammatory response. Granulocyte-macrophage colony-stimulating factor?GM-CSF?, a new high-efficiency molecular adjuvant, is a kind of immune regulating factor that activate the dendritic cell and enhance ability of antigen presentation. VP2 protein, a major structural protein of infectious bursal disease virus?IBDV? and host protective antigen, is one of target gene of IBD DNA vaccine. In this study, we optimized and synthesized Gallus Akirin2 and Gallus GM-CSF gene, and evaluated their regulating immunity effect on IBD VP2 DNA vaccine, the results reflected an important academic value and application prospect.1. According to the published gene sequences of IBDV VP2?AF508177?, Gallus Akirin2?NM₀01193595? and Gallus GM-CSF?EU939770.1?, we optimized and synthesized their coding region, connected by connecting peptide and inserted into p Tri Ex-4 to obtain VP2, Akirin2 and GM-CSF protein co-expression recombinant plasmids: p VP2-Akirin2-GM-CSF. Then the p VP2-Akirin2-GM-CSF was used to transfect into 293 T cells and analyzed by SDS-PAGE and Western blot. As expected, the three calculated protein were approximately58 k Da, 36 k Da and 22 k Da, and could be specifically recognized by specific antibodies, respectively. Meanwhile, we cloned other five recombinant plasmids: p VP2, p Akirin2, p GM-CSF, p VP2-Akirin2 and p VP2-GM-CSF.2. The recombinant plasmid p VP2 was used to construct recombinant baculovirus Bac-VP2 by homologous recombination with Bac Magic-3 DNA in Sf9 cells. The result of immunofluorescence assay showed specific fluorescent signal by IBD positive serum. Then we extracted proteins of infected Sf9 cells and purified through Ni-chelating affinity chromatography to obtain His-VP2 fusion protein. The protein was used in ELISA to develop differential diagnostic methods. Through experiments, ELISA methods had higher sensitivity and repeatability by IBD standard serum and higher specificity by other chicken disease positive sera, like Mareker's Disease?MD?, Avian Leukosis?AL?, Reticuloendotheliosis?RE?, Avian Influenza?AI? and Newcastle Disease?ND?.3. The recombinant plasmid p VP2-Akirin2-GM-CSF was injected into 14 days SPF chickens as DNA vaccine, and boosted with same methods at 28 days. 14 days later, the SPF chickens were challenged by IBDV of BC6-85 strain, and autopsied 4 days after the virus challenging, the result showed that this DNA vaccine could provide protection against IBDV with 85%, while the p VP2 group protective rate was 65%, the p VP2-Akirin2 group protective rate was 75%, and p VP2-GM-CSF group protective rate was 70%, respectively. Sera were collected at 14, 28, 42, 46 days old to detect VP2 antibodies level and neutralizing antibodies against IBDV. The results showed that antibodies' level continued rising after immunization. At age of 42 days, the p VP2-Akirin2-GM-CSF induced VP2 antibodies level?S/P? with 1.106, which were no significant higher than p VP2-Akirin2?0.973?, but very significant higher than the p VP2?0.771? and the p VP2-GM-CSF?0.836?. Meanwhile, the p VP2-Akirin2-GM-CSF induced neutralizing antibodies against IBDV with 1:4298, which were very significant higher than the p VP2?1:2057?, the p VP2-Akirin2?1:3514? and the p VP2-GM-CSF?1:2557?. Moreover, the peripheral blood lymphocyte proliferating ability was significant higher than other groups at 14 days after boosted immunization. At last, the concentration of IFN-?, IL-2, IL-4 and IL-6 separated from 42 days old chickens' sera of injected with p VP2-Akirin2-GM-CSF were detected by ELISA. The results showed that the concentration of IFN-?, IL-2 and IL-6 were up-regulated, and the the concentration of IL-4 was down-regulated. The research showed that the p VP2-Akirin2-GM-CSF could induce stronger humoral immunoresponse and cellular immunoresponse.