Establishment And Preliminary Protective Effects Evaluation Of Yeast Surface Display Of Infectious Pancreatic Necrosis Virus VP2 Protein

Infectious pancreatic necrosis?IPN?is one of the most serious viral diseases in salmon and trout aquaculture,especially in salmon fry and post-smolt.The epidemic spreads throughout Asia,Europe and the United States.In 1986,the first epidemic of IPN occurred in Shanxi Province,China.And then outbreaks of IPN were reported in many regions of China.In 2013,an infectious pancreatic necrosis virus?IPNV?strain was collected in a rainbow trout farm in Yunnan Province,China,where outbroke a severe IPN disease.The complete genomic sequence of this virus was confirmed and submitted to the GenBank database and named as ChRtm213?accession no.KX234591?.A considerable difference was found between the ChRtm213 isolate and other reported Chinese isolates in serotype.In 2016,another IPNV strain was also isolated in Sichuan Province,China.Based on these results,we cannot afford to ignore the importance of preventing this viral disease.In this study,a yeast display system was established to express the VP2 gene of the ChRtm213 strain.The VP2 gene was amplified and inserted into pYD1 vector using Kpn I and Not I restriction sites,and the reliable plasmid was named as pYD1-VP2.The Saccharomyces cerevisiae strain EBY100 was transformed with pYD1-VP2 plasmid using electroporation,and then incubated with YNB-CAA medium containing 2%galactose to induce the expression of VP2 protein on yeast cell surface.The VP2 expression was confirmed by Western blotting and immunofluorescence assay.The Western blotting results showed that a specific VP2protein was observed in the induced EBY100/pYD1-VP2 cell.The immunofluorescence assay result showed that the EBY100-pYD1-VP2 cell presented a specific green fluorescence on the cell surface,and the fluorescence intensity was positively correlated with induction time.These results indicated that the VP2 protein was successfully displayed on the yeast cell surfaces.The successful display of VP2protein on the yeast surface laid a foundation for live oral vaccine development against IPN disease.In order to evaluate the immunological efficacy of the oral vaccine,an oral immunoassay was developed on rainbow trout.We.The oral yeast vaccine,designated EBY100-pYD1-VP2,was delivered orally to rainbow trout?Oncorhynchus mykiss?.EBY100-pYD1(1×10¹⁰ CPU/ml)was used as empty vector control and PBS was used as blank control.At the 30 days post-vaccination?d.p.v.?,then challenged with?IPNV100?l 100TCID50/ml?by intraperitoneal injection.At the30 days after challenge,the expression of VP1 and VP4 genes were determined by Quantitative Real-time PCR?RT-qPCR?.The rainbow trout not challenged were set as negative control.The results of viral load analysis showed that the expression levels of VP1 and VP4 genes in the immunized group were significantly lower than those in the blank group at the 30?d.p.v??P<0.05?.The expression levels of VP1gene in the empty vector group and the blank group were 147-fold and 181-fold compared with the immunized group.The expression levels of VP4 gene were170-fold and 189-fold.The results showed that the average viral load of was significantly reduced by oral inoculation compared with the blank group and the empty vector group?P<0.05?.On the 60?d.p.v.?,the neutralizing antibody titer was determined by the Neutralizing antibodies test.The results of the neutralizing antibody test showed that the average neutralizing antibody titer in the immunized group was 30.2,which was higher than that in the negative group which was not challenged?P<0.05?.The expression levels of IgM,IgT,CD4 and CD8 genes in the head kidney,spleen and hindgut tissues were analyzed by RT-qPCR at the 7?d.p.v.?and 14?d.p.v.?.The results of RT-qPCR showed that at the 7?d.p.v.?,the expression levels of IgM and IgT in the head kindy were 6.9-fold and 5-fold higher than that in the blank group,in the spleen were 7.9-fold and 5.6-fold,in the hindgut were 8.6-fold and 5.8-fold.compared with the blank group,the expression of IgM and IgT genes in the kindy,spleen and hindgut was significantly significantly upregulated?P<0.05?.At the 14?d.p.v.?,the expression levels of IgM and IgT in the spleen were 7.4-fold higher than that in the blank group,The expression of IgM and IgT genes in the spleen of the immunized group was significantly upregulated?P<0.05?.The results of RT-qPCR showed that at the 7?d.p.v.?,the expression levels of CD4 and CD8 in the head kindy were 5.3-fold and 8.2-fold higher than that in the blank group,in the spleen were 7.1-fold and 8.7-fold,in the hindgut of the immunized group were8.6-fold and 7.2-fold,Compared with the blank group,the expression of CD4 and CD8 genes in the head kidney,spleen and hindgut was significantly upregulated?P<0.05?.at the 14?d.p.v.?,the expression levels of CD4 in the spleen and hindgut were 11-fold and 8.2-fold higher than that in the blank group,Compared with the blank group,the expression of CD4 genes in the spleen and hindgut was significantly upregulated?P<0.05?.Based on the analysis of viral load,neutralizing antibody titer and immune-related gene expression,oral EBY100-pYD1-VP2 immunization significantly reduced the IPNV load in rainbow trout and produced higher levels of neutralizing antibody valence induced a significant increase in the expression of immune-related genes.This study demonstrates that the IPNV vaccine constructed by the yeast surface display system can significantly reduce the IPNV viral load in rainbow trout,and can stimulate the non-specific immunity and specific immune response of rainbow trout,laying a foundation for the development of a new oral live vector vaccine against IPNV.Based on the analysis of viral load,neutralizing antibody titer and immune-related gene expression,oral EBY100-pYD1-VP2immunization significantly reduced the IPNV load in rainbow trout and produced higher levels of neutralizing antibody Valence induced a significant increase in the expression of immune-related genes.This study demonstrates that the IPNV vaccine constructed by the yeast surface display system can significantly reduce the IPNV viral load in rainbow trout,and can stimulate the non-specific immunity and specific immune response of rainbow trout,laying a foundation for the development of a new oral live vector vaccine against IPNV.