Preparation And Evaluation Of The Recombinant Oral Bacillus Subtilis Subunit Vaccine Of Grass Carp Reovirus

Grass carp(Ctenopharyngodon idella)is one of the main varieties of freshwater aquaculture in China,with important economic value.However,Grass Carp Reovirus(GCRV)causes a high mortality rate of Grass Carp hemorrhagic disease,which caused huge losses to Grass Carp breeding industry.Epidemiological studies have shown that GCRV II is now the prevailing genotype,so it is necessary to develop an effective and practical GCRV II vaccine.However,most of the vaccines are injectable,which have practical difficulties and high cost.The development of oral vaccine is a good strategy to solve the above problems.In this study,an oral subunit vaccine of Bacillus subsubis was prepared with coat protein VP56 of grass carp reovirus type II as the research object,and the immune protection effect of the vaccine was evaluated by oral immunization.This study is divided into three parts:screening of potential epitopes of VP56 protein,preparation of oral vaccine of Bacillus subtilis and in vivo efficacy detection.1.Screening of potential epitopes of VP56 proteinWe used DNASTAR software to analyze the epitopes of VP56 protein.We divided it into 4 fragments,amplified 5 sequence genes including the full length of VP56 and the 4 fragment respectively,and constructed these 5 sequence genes on p GEX-4T-1 plasmid to express and purify these 5 proteins.Five proteins were first combined with anti-GCRV serum for ELISA.The result showed that VP56-2 and VP56-4 were significantly different from other groups in ELISA experiment,indicating that these two fragments had a good ability to bind to antibodies.VP56-2and VP56-4 proteins were injected into grass carp to obtain anti-VP56-2 and VP56-4serum,and the anti-VP56-2 and VP56-4 serum were used to conduct antibody neutralization experiment,and the potential epitopes of VP56 were screened.The results showed that when the antiserum dilution ratio was 1:5000,a large number of cells died in VP56-4 group,while the dead cell in VP56-2 group was significantly less than that in VP56-4 group,so the anti-VP56-2 serum had a good anti-GCRV ability.The VP56-2 was the potential antigen site of VP56.2.Preparation of oral subunit vaccine of B.s-CotC-VP56-2We first constructed VP56-2 fragment on plasmid p HT304,then extracted the DNA of Bacillus subtilis WB600.We amplified the spain-anchored protein CotC sequence,and constructed CotC into recombinant plasmid p HT304-VP56-2 by one-step cloning method.The expression of VP56 protein were detected by sds-page and Western blotting in spore capsid.After 24h induction,the spore fluid was fixed and incubated with anti VP56 mouse serum and anti-mouse Cy3-labeled Ig G of goat,respectively.The expression of VP56-2 protein in spore was observed under fluorescence microscope.Then about 10~6CotC-VP56-2 spores were re-suspended and incubated with anti-VP56 mouse serum and FITC-conjugation goat anti-mouse Ig G,respectively.Flow cytometry was used to count the rate of VP56-2 expression in the spores.The results of SDS-PAGE and Western blotting showed that VP56-2 protein was expressed in the outer shell protein of Bacillus subtilis.Immunofluorescence showed that VP56-2 protein was expressed on the surface of spore.The expression rate of VP56-2 protein in Bacillus subtilis was 82.22%by flow cytometry.In conclusion,recombinant Bacillus subtilis B.s-CotC-VP56-2 can successfully express VP56-2 protein on the surface of the spores,proving that it has the possibility of becoming an oral subunit vaccine.3.In vivo effect detectionIn order to test the immune effect of B.s-CotC-VP56-2,400 fish were divided into 4 groups:Control,B.Subtilis,B.s-CotC and B.s-CotC-VP56-2,with 100 fish in each group.On days 1,3,7,14,21,28,30,32 and 36,four fish were randomly selected from each group for sampling and detection.We determined the protective effect of each experimental group by means of protection rate analysis,quantitative detection of specific antibodies,intestinal colonization analysis and quantitative immune-related genes.The results showed that the protective rate of B.s-CotC-VP56-2 group(56%)was significantly higher than that of the other groups,and the quantitative detection results confirmed that the specific antibody level of B.s-CotC-VP56-2 group was higher than that of the other groups.The serum C3 content,lysozyme and TSOD activity in B.s-CotC-VP56-2 group were also increased.There were significant differences with other groups.After challenge,the immune genes IL-1β,TNF-α,IFN1 and MHC II of B.s-CotC-VP56-2 were significantly up-regulated by q RT-PCR,and combined with the results of viral load detection.It was found that B.s-CotC-VP56-2 could inhibit the replication of GCRV.According to pathological section analysis.The pathological changes of B.s-CotC-VP56-2 group were the least,while the pathological changes of B.subtilis and B.s-CotC groups were slightly lighter than those of the control group,which also proved that Bacillus subtilis could enhance the immune capacity of grass carps,but did not have the ability to resist the invasion of GCRV.According to the results of in vivo detection,oral administration of B.s-CotC-VP56-2 is better than other groups.The advantages of this method are that it can effectively improve the protection rate of grass carp infected with GCRV II,increase the expression of specific antibodies,effectively activate the immune pathway,protect tissues and organs,and inhibit virus replication.In addition,Bacillus subtilis also enhances the immune capacity of grass carp,and its multi-directional protective effect makes grass carp more resistant to GCRV II virus infection,thus achieving better results,providing a new effective strategy for the development of grass carp anti-GCRV vaccine.