Evaluation Of The Protective Immunity Induced By Avian Reovirus σC Protein Expressed In Pichia Pastoris

Avian reovirus infection is an important immunosuppressive disease in poultry industry which iscaused by avian reovirus, it can cause viral arthritis and tenosynovitis in chickens and it also relates withchicken’s respiratory disease, enteric diseases, small syndrome, temporary digestive disorders andmalabsorption. In order to develop a subunit vaccine for ARV prevention, the σC gene of ARV wasamplified from ARV S1133strain using σC specific primers and cloned into the pPIC9K vector. Therecombinant plasmid pPIC9k-σC was identified, linearized with Sal I and then transformed into P.pastoris strain GS115by electroporation. Transformants were selected on MD plates and then on YPDplates containing G418and confirmed by PCR analysis. SDS-PAGE assay demonstrated that σC proteinwas expressed and secreted into the culture medium which is existed as monomer and trimer. Theexpression product amounted to42mg/L of culture. The recombinant σC protein showed goodantigenicity in western blot using anti-σC polyclonal antibody and anti-S1133polyclonal antibody.In order to improve the expression level of the recombinant P. pastoris strain, the P. pastoris hoststrain, the copy number integrated into the Picher genome and the fermentation conditions wereoptimized. In this study, Pichia strain KM71and GS115were selected as the host strains fortransformation, and the effects of Mutsand Mut+phenotype for σC protein expression were compared.To analysis whether the improvement of the gene copy number could enhance the σC protein expressionlevel, the recombinant plasmid pPICZαC-σC was developed and transformed into pPIC9K-σC/GS115P.pastoris strain. The fermentation conditions including pH and temperature were also optimized. Theresults revealed that Mut+is better than Muts; improving gene copy number could enhance the σCexpression level; the optimal pH is7.0and the optimal temperature is30℃. Under the optimizedconditions, the σC protein level can amount to540mg/L. The optimization of the expression conditionslaid the foundations for the fermentation production in the industry.The recombinant σC protein was adjuvanted and injected into3-week-old SPF chickens with80μgσC protein per chicken. Boost immunization was conducted with the same dose four weeks after thefirst immunization. At four weeks after the first immunization, another immunized group waschallenged with ARV S1133strain. The results revealed that the antibody titers increased sharply afterthe second injection and reached to the high level platform. The antibody level was5000~7000between2weeks and17weeks after boost immunization and decreased slightly. Serum neutralization testshowed that the immunized serum can resist the ARV S1133infection and neutralization titer can be ashigh as1:1000. The challenging experiment showed that the vaccine could inhibit virus replication andclear the virus. To sum up, the σC protein which could secretory expressed in Pichia Pastoris showedgood antigenicity and could also induced protective immunity. This study laid the foundations fordiagnosis and subunit vaccine research of ARV S1133.