Simultaneous Expression Chicken Anemia Virus VP1 And VP2 In Pichia Pastoris, Hight-Density Fermentation Of Recombinant Pichia Pastoris And Immunological Protection Assay

Chicken anemia virus(CAV) is the only member of the genus of Gvrovirus of the family Circoviridae.It is a significant immunosuppressive pathogen of chickens which results in the bone marrow aplasia,thymus and bursal lesion.The viral genome is a single-stranded DNA that is circular and covalently linked and consists of approximately 2300 bp in its replicative form.There are three open reading frames(ORF1,ORF2 and ORF3) with 648 bp,363 bp and 1350 bp in size,respectively.VP3 can induce the apoptosis. VP1,capsid protein(54 kD) is the major structural protein.VP2(24 kD) involves in the conformation development of VP1.VP1 and VP2 proteins induce to generate the neutral antibody production,which are the best candidate for the recombinant subunit vaccine development.In this study,we constructed two recombinant eukaryotic expression plasmids pPIC9K-VP1 and pPIC9K-VP2.pPIC9K-VP1 and pPIC9K-VP2 were used for co-transduction into Pichia pastoris strain GS115.Based on the identification of PCR and induced expression by Methanol,we gamed the successful recombinant strain which could co-express protein CAV VP1 and VP2.The CAV VP1 protein could secrete into supernatant,however,the CAV VP2 could not release until the cell was to break into pieces. The results of Dot-ELISA and Western blot assays indicated that the non-structural protein VP2 played a critical role to cooperation of the tertiary,structure formation of VP1 protein. The CAV VP1 protein possessed the conformational epitope,the production of the neutral antibody of CAV was determined by the interaction of the VP1 and VP2.In this study,the recombinant Pichia pastoris strain(GS115 pPIC9K and CAV VP1-VP2) was obtained and the CAV VP1 and VP2 were successfully co-expressed in a GS 115 cell.The recombinant Pichia pastoris strains.GS115 pPIC9K and CAV VP1-VP2 were used for the small scale high-density fermentation.After the optimization of conditions of the fermentation,we gained the best condition for the fermentation as followed:30℃,pH 5.0,DO value 40%,Methanol added as 7.5ml/L·h.The OD₆₀₀ value could reach 1200 or higher than 1200 after 72h.,the expressed protein was stored at -20℃.The production of the fermentation of five liter medium was to break into pieces by hyperpressure. Spectrophotometric-quantification and SDS-PAGE TLC showed that the yield of the VP1 and VP2 protein is 34.2mg/ml which was account for 30%in the production of the total proteins.This assay indicated that VP1 and VP2 proteins were high-efficiently expressed in the small scale high-density fermentation.The superiorities of this expression system were showed conspicuous because the VP1 and VP2 proteins with high yield and stability were obtained in the fermentation using FBS.The VP1 and VP2 proteins obtained from the small scale fermentation was purified and concentrated.The concentrated proteins mixed with the ontanide ISA50 adjuvant,the safety and immunity of this recombinant subunit vaccine was evaluated as followed.The vaccine was serially diluted.The 10-day-old SPF chickens were randomly divided into five groups(5 chickens in each group),every group was injected vaccine with different dilutions.The histopathologic results obtained 21 days post vaccination indicated that this recombinant subunit vaccine was safe.Four groups(10 chickens in each group) vaccinated with different diluted vaccine.The sera obtained both before vaccination and post vaccination were used for evaluated by ELISA,and the results showed that the antibody was detected only in the vaccination groups while not in the control group.Besides,the titer of the antibody increased in the whole process of this assay after vaccination.The IFA result showed the antibody obtained from the vaccinated chickens reacted strongly with the MDCC-MSB1 cells inoculated the CAV.After three weeks of vaccination,chickens vaccinated or not were changed with high virulent CAV.21 days post change,the histopathologic results showed that the chickens vaccinated were normal while the conspicuous histopathologic changes observed in the not vaccinated groups.The PCR results showed that the positive percentage was 7%in the vaccinated group.the percentage of not vaccinated group was 100%.This study showed that this recombinant subunit vaccine were safe and possessed good immunity.