Establishment And Immune Efficacy Of Yeast Surface Display System For Spring Virernia Of Carp Virus Glycoprotein

The spring virernia of carp?SVC?,caused by the spring viremia of carp virus?SVCV?,is a serious disease of carp with high mortality.Office International Des Epizooties?OIE?lists SVC as an important disease that must be declared.SVCV was defined as category I animal diseases in China in 2008.This disease usually occurs in the spring with the characteristics of a wide range of epidemics,high morbidity and mortality,and brings huge economic losses to fish farming around the world every year.Currently,there is no effective treatment of SVC.For the prevention and control of viral diseases,vaccines have always been the most ideal choice.Oral vaccine is currently the most popular fish vaccine type,which not only can solve the drawbacks of heavy workload,time and effort,but also can be used for large-scale immunization.In this study,SVCV glycoprotein?G?was displayed on surface of Saccharomyces cerevisiae EBY100 to construct an oral vaccine.The young carp?10±2g?was orally immunized.The contents and results of the study are listed as follows:1.In this study,open reading frame?ORF?of glycoprotein?1530 bp?was amplified through RT-PCR by using RNA extracted from SVCV as template.The SVCV G ORF was cloned into pYD1 vector to construct a recombinant plasmid pYD1-G.The pYD1-G was electro-transformed into the competent yeast cells EBY100.The transformed cells were spread on YNB-L?Leucine?Medium and induced by 2%glucose.The expression of G protein on the surface of Saccharomyces cerevisiae was detected by cell immunofluorescence and flow cytometry.The immunofluorescence results showed that specific red fluorescence could be observed from induced EBY100-pYD1-G,and the portion of fluorescent yeast cells increased with the prolong of induction time.Using Image J software to analyze the immunofluorescence microscopic field?n=10?,the average proportion of positive yeast cells at 0,24 h,48 h and 72 h induction was 8.38%,24.20%,55.30%and 72.41%,respectively.The proportion of positive yeast between the groups was significantly different?P<0.05?.The flow cytometry results showed that the fluorescence intensity of yeast cells was proportional to the induction time.At 0,24 h,48 h and 72 h,the average fluorescence intensity of yeast cells was 2112,7086,10236,10554,respectively.There was no significant difference in the fluorescence intensity of yeast cells between 48h and 72 h,which basically reached a stable state.Therefore,the optimal induction time for yeast surface display was selected for 48h-induction.2.A large number of recombinant yeasts expressing SVCV G protein?1×10⁹CPU/ml?were prepared to immunize carp?10±2 g?,100?L per oral administration.The carp were divided into 4 groups,the EBY100-pYD1-G group?Single immunization and Multiple immunization?and EBY100-pYD1 group?Single immunization and Multiple immunization?.The single immunization group refers to continuous immunization for 7days and the multiple immunization group refers to immunization for 2 times,interval 7d,continuous immunization for 7 days.The head kidney and spleen of carp were collected on the 7,14,21 and 28 days post vaccination?d.p.v.?,and CD4,TNF-?and Ig-T genes were determined by Reverse Transcription Quantitative Real-time PCR.The neutralizing antibody titer of each group was detected by EPC on the 42 d.p.v.On the 30 d.p.v.,the immunized carp were challenged by intraperitoneal injection with 10⁶.28.28 TCID₅₀/mL of SVCV at concentration of 100 TCID50/mL?50?L/fish?,and the death of carp was continuously recorded for 21 days to calculate the relative protection of oral vaccine.The results of genes expression showed that,the expression of CD4,TNF-?and IgT genes were up regulated after oral administration of recombinant yeast EBY100-pYD1-G.The expression level of CD4 gene peaked at 21 d.p.v.On 28 d.p.v,the expression of CD4 in the EBY100-pYD1-G group was 3.26-fold and 3.19-fold of that in the single immunization EBY100-pYD1-G group in the head kidney and spleen,respectively?P<0.05?.The expression level of TNF-?gene peaked at 21 d.p.v.At this time point,the expression of TNF-?gene in EBY100-pYD1-G group was significantly increased in the head kidney,which was 3.29-fold of that in the single immunization group of EBY100-pYD1-G,?P<0.05?.The Ig-T gene reached its maximum at 14 d.p.v.At this time point,the gene expression of multiple immunization EBY100-pYD1-G was significantly increased in the head kidney and spleen,which was 3.18-fold and 3.07-fold of that in the single immunization EBY100-pYD1-G,?P<0.05?.Serum neutralizing antibody titration results showed that the average titers of neutralizing antibodies in the single immunization group of EBY100-pYD1-G and the multiple immunization EBY100-pYD1-G group were 31.9and 40.6,respectively.There was no neutralizing antibody in the serum of EBY100-pYD1immunized group,and significant difference were observed ach of the groups?P<0.05?.The challenge experiments showed that the relative protection rates of single immunization EBY100-pYD1-G and multiple immunization EBY100-pYD1-G were 37.14%and 43.24%.This results indicated that immunized EBY100-pYD1-G recombinant yeast could significantly reduce the mortality of carp?P<0.05?,and the immune effect of multiple immunization groups was significantly better than single immunization group?P<0.05?.In summary,the SVCV live yeast oral vaccine can stimulate the carp to produce specific and non-specific immune responses and has significant protective effects against SVCV infection.It could be likely used as a novel oral vaccine for fish against SVCV.