Expression Of IBDV VP2-VP5-IgY Fc Fusion Protein In Pichia Pastoris And Research Of Immunogenicity

Infectious bursal disease(IBD)is an acute and highly contagious disease caused by IBD virus(IBDV).After the virus infection,IBDV mainly leads to damage in lymphoid organs,resulting in immunosuppression which leads to vaccination failure and susceptibility to other infections.VP2 protein,the major structural protein,is the major host-protective antigen of IBDV,which contains the major neutralization sites and shows serotype specificity.VP5 protein is inessential for virus replication in vitro but bears important responsibility for pathogenesis and dissemination.VP5 protein has effective immunogenicity and induces the production of high titer polyclonal antibodies.In addition,it was found that when a mammalian Ig G Fc fragment was ligated to a segment of a gene,the recombinant protein was given characteristics of the antibody,thereby enhancing the half-life period of immunogen and increasing the efficiency of antigen capture.In avian species,IgY is similar to mammalian IgG in terms of structure which effectively enhances the immune response induced by the antigen,however,there is little research on the function of Ig Y Fc.Pichia pastoris,a standard expression system,can express foreign proteins with high efficiency.This eukaryotic expression presented several advantages,such as easy cultivation,high yield,precise post-translational modifications,and minimal interference of native proteins.Adjuvants play an important role in the immune response process,it can increases the adaptability to specific antigens by activating the innate immune system,thereby establishing a high and long-term immune response.In our previous study,we found that Taishan Masson Pine Pollen Polysaccharide(TPPPS)is an immune adjuvant that has a good immunomodulatory effect on inactivated and subunit vaccines.In this study,P.pastoris GS115 eukaryotic expression system was used to express fusion protein containing VP2-VP5 of IBDV and the chicken IgY Fc.In addition,we selected TPPPS as an adjuvant investigated its immunomodulation effects on on immune responses.This study provided theoretical basis and technical support for the IBDV subunit vaccine and immune prevention.1 Construction of recombinant plasmid of pPIC9-VP2-VP5-FcThree pairs of specific primers were designed according to the VP2 and VP5 gene sequences of IBDV(GenBank accession number: AY907014.1 and EF101893.1)and chicken IgY Fc gene(GenBank accession number: X07174).The chicken IgY Fc and VP2 and VP5 of IBDV were linked by overlapping PCR technology.The VP2-VP5-Fc fragment was linked into the pMD18-T vector and validated by sequencing,then the recombinant plasmid was digested with EcoR I and Not I restriction enzyme.The digestion product was linked to pPIC9 plasmid which was digested with same restriction enzyme and validated by sequencing.The recombinant expression vector pPIC9-VP2-VP5-Fc was transformed into P.pastoris GS115.The recombinant positive transformant was selected through RDB media,and then validated by PCR and sequencing.P.Pastoris transformed with blank pPIC9 plasmid was used as a negative control.2 Expression and identification of the recombinant protein in P.pastorisMethanol induces the protein expression.The culture supernatants were harvested by centrifugation at 24,48,72 and 96 h after methanol induction,and identified by SDS-PAGE and Western blot analysis.After 24 h of cultivation,the protein was detected in the supernatant apparently,and the maximum protein concentration(9.6,8.3,and 13 mg/L)occurred at 72 h individually.Single-protein bands with a molecular weight of 25.63,49.35 and 67.09 kDa was detected in the result of SDS-PAGE after purification.After Western blot analysis,we observed a single reaction band corresponding to the band in SDS-PAGE.3 Immune-enhancing effects of TPPPS on the recombinant fusion proteinThe purified recombinant VP2–VP5–Fc protein was mixed with TPPPS at a specific ratio.A total of 240 1-day-old male specific pathogen-free(SPF)white leghorn chickens were randomly divided into six sterilized isolators on average.All chickens in groups I–VI were subcutaneously inoculated with VP2-VP5-Fc +TPPPS,pure VP2-VP5-Fc,VP2-Fc,VP2,commercial vaccine,and PBS at 9 days old.At 0,7,14,21,28,35,42,and 49 days after vaccination(dpv),Immune parameters in serum of each group were detected by ELISA assay,flow cytometry,and MTT assay,respectively.and the following aspects were mainly examined: serum antibody titer,interleukin-2(IL-2),interleukin-4(IL-4)and interferon-?(IFN-?),CD4+ and CD8+ lymphocytes levels in the peripheral blood and the T lymphocyte transformation rate.Three weeks after the vaccination(21 dpv),30 chickens of each group were placed in a new isolator and intranasally challenged with 2,000 median embryo lethal dose(ELD50)of the virulent IBDV.Clinical symptoms and survival status of the chickens were monitored for 7 days after challenge.The results showed that the effect of the recombinant protein VP2-VP5-Fc of IBDV was better than that of other groups.In addition,TPPPS used as adjuvant exhibited a big enhancement for the immune response of the recombinant subunit.Therefore,this study provided technical and theoretical supports for the development of IBDV subunit vaccine and the application of TPPPS used as adjuvant.