Construction And Immune Efficacy Of The Bivalent Gene OmpN2-OmpF Subunit Vaccine And Oral Vaccine Against Edwardsiella Ictaluri And Yersinia Ruckeri In Channel Catfish

Aquaculture vaccine,as a safer and more effective method to prevent and treat fish bacterial diseases instead of antibiotics and chemical drugs,has attracted extensive attention in the world.Combined vaccine has become the research and development direction of vaccine in the world because of its advantages of preventing various diseases,reducing vaccination frequency and simplifying immunization procedures.Edwardsiella ictaluri and Yersinia ruckeri,as the pathogens of two common bacterial diseases of channel catfish(Ictalurns Punctatus)in recent years,had caused serious harm to channel catfish even the entire aquaculture healthy development.In this study,firstly,on the basis of molecular cloning,bioinformatics analysis,prokaryotic expression and immunogenicity analysis,the potential of outer membrane protein OmpN2 of E.ictaluri and outer membrane protein OmpN2 of Y.ruckeri were studied as a candidate antigen for the corresponding vaccines.Secondly,the bivalent fusion gene N2-F was constructed by adding linker sequences,enzymatic ligation and the fusion gene technology,then the bivalent genetic subunit vaccine N2-F(fusion protein N2-F)was constructed and prepared by molecular cloning and prokaryotic expression.Thirdly,the bivalent genetic oral vaccine was constructed using natural polymer compounds including sodium alginate and chitosan as coating materials.Fourthly,the healthy channel catfish were intraperitoneally injected and orally vaccinated using N2-F bivalent genetic subunit vaccine and bivalent genetic oral vaccine respectively,the immunoprotection efficiency and mechanism were evaluated and analyzed by the specific antibody levels,non-special immune indices,immune-related genes expression and relative percent survival(RPS),which provided important references for the development of safe and effective combined vaccine and the prevention and treatment of channel catfish E.ictaluri and Y.ruckeri diseases.This study included the following contents:1.Molecular cloning and bioinformatics analysis of OmpN2 and OmpF genesThe comprehensive molecular properties analysis and antigen epitope prediction were conducted through the molecular cloning and bioinformatics analysis on OmpN2 and OmpF gene sequences and deduced amino acid sequence,the results showed that OmpN2 and OmpF gene sequences were 1122 bp and 1095 bp in length respectively,which encoded 373and 364 amino acid sequences respectively.They both possessed the gram_neg_porins conserved domains,respectively belonged to the outer membrane proteins of E.ictaluri and Y.ruckeri with 9 and 12 potential antigenic determinant domains.The similarity of their amino acid sequences was 65.96%,and the 3D structure prediction indicated that their spatial conformation were both trimers and they were more than 60%similar with the same template,which suggested that there were some similarity on their structure and function and they could provide the cross-protection for each other as the vaccine candidate antigens.2.Prokaryotic expression and immunogenicity analysis of OmpN2 and OmpFBy prokaryotic expression and the removing of corresponding signal peptides,the recombinant proteins rtOmpN2 and rtOmpF were successfully constructed with about 56kDa and 55 kDa respectively.The results of bacteria surface staining and indirect immunofluorescence showed that E.ictaluri and Y.ruckeri could specifically bind to the rabbit anti-rtOmpN2 antibody and rabbit anti-rtOmpF antibody,respectively and showed the positive reaction signals,indicating that OmpN2 and OmpF proteins were the bacteria surface antigens of E.ictaluri and Y.ruckeri,respectively.The results of Western blotting showed that rtOmpN2 and rtOmpF could be recognized and reacted with rabbit anti-E.ictaluri and rabbit anti-Y.ruckeri antibodies respectively,indicating that OmpN2 and OmpF proteins were not only one of the antigens of E.ictaluri and Y.ruckeri,but also possessed good immunogenicity and could be used as candidate antigens for genetic engineering vaccines.3.Construction,molecular cloning,prokaryotic expression and immunogenicity analysis fusion gene N2-FThe fusion gene N2-F was successfully constructed a size of 2133 bp by adding Linker sequence and BamH I restriction site ligation method,then the recombinant fusion protein N2-F were successfully expressed with about 96 kDa using prokaryotic expression.The results of Western blotting indicated that N2-F could specifically bind to the rabbit anti-6×His antibody,rabbit anti-E.ictaluri and rabbit anti-Y.ruckeri antibodies,respectively and showed the single and obvious bands,indicating that the fusion protein N2-F was successfully expressed,simultaneously possessed the protein conformation of OmpN2 and OmpF that could be recognized by corresponding antibodies.The results of bacteria surface staining analysis showed that E.ictaluri and Y.ruckeri could specifically bind to the rabbit anti-N2-F antibody and showed the positive reaction signals respectively,indicating that fusion protein N2-F had the immunogenicity of OmpN2 and OmpF simultaneously,which could be used as the candidate antigens to construct the bivalent genetic subunit vaccine against E.ictaluri and Y.ruckeri infection simultaneously.4.Immune effect analysis of bivalent genetic subunit vaccine in channel catfish.Compared with monovalent vaccine rtOmpN2 and rtOmpF,the bivalent subunit vaccine N2-F significantly(P<0.05)increased the serum corresponding antibody levels,the serum nonspecific immune indexes including lysozyme activity,C3 activity,total protein content,and SOD activity,the immune related genes expression in head kidney and spleen in channel catfish.The RPS of channel catfish in N2-F vaccine group against E.ictaluri and Y.ruckeri infection was 76.67%and 82.71%,which was respectively higher than rtOmpN2(RPS was73.33%)and rtOmpF(RPS was 75.86%),indicating that N2-F vaccine not only resist E.ictaluri and Y.ruckeri infection respectively,but also provided higher and better immuneprotection efficiency.5.Construction,condition optimization and physicochemical properties analysis of of bivalent genetic oral microsphere vaccineOn the basis of the condition optimization of sodium alginate-chitosan composite microsphere using response surface method(RSM),the optimal construction condition of N2-F composite microsphere were sodium alginate concentration with 1.58%,water/oil phase ratio with 4.0:6.0,CaCl₂ concentration with 8.60%and chitosan solution concentration with 0.82%,in this condition,the encapsulation efficiency of N2-F composite microsphere showed as 93.26%and the particle size showed as 4.35±1.08?m.The results of physical and chemical properties analysis of composite microspheres showed that N2-F composite microspheres were sensitive to the temperature,pH and ionic strength of the solution to various degrees.The swelling ratio was increased in the solution with higher temperature,alkaline environment and ionic strength.The release performance of composite microspheres was analyzed in simulated gastric and intestinal solution in vitro,the results indicated that N2-F microsphere in simulated gastric juice(acidic environment)released slowly,while in the simulated intestinal fluid(alkaline environment)released fast,which suggesting that the N2-F microspheres not only protected purpose protein antigen from being destroyed in fish stomach,but also guaranteed the purpose antigen to be released for absorption in fish intestinal.The results of Western blotting analysis showed that the protein released from composite microspheres was the target protein antigen and maintained good immunogenicity.Safety test showed that the compound microspheres were safe for fish and could be used as oral vaccine in fish.6.Immune effect analysis of bivalent genetic oral microsphere vaccine in channel catfish.Compared with monovalent oral microsphere vaccine rtOmpN2 and rtOmpF,the bivalent oral vaccine N2-F significantly(P<0.05)increased the intestinal mucus and serum corresponding antibody levels,the nonspecific immune indexes including lysozyme activity,C3 activity,total protein content,and SOD activity,the immune related genes expression in head kidney and spleen in channel catfish.The RPS of channel catfish in N2-F oral vaccine group against E.ictaluri and Y.ruckeri infection was 73.33%and 79.31%,which was respectively higher than rtOmpN2 oral vaccine(RPS was 63.33%)and rtOmpF oral vaccine(RPS was 65.52%),indicating that N2-F oral vaccine not only resist E.ictaluri and Y.ruckeri infection respectively,but also provided higher and better immuneprotection efficiency.In conclusion,on the basis of fusion gene N2-F construction,the bivalent genetic subunit vaccine and oral microsphere vaccine were successfully constructed.The healthy channel catfish were intraperitoneally injected and orally vaccinated using the N2-F bivalent genetic subunit vaccine and bivalent genetic oral vaccine respectively,and the immunoprotection efficiency was comprehensively evaluated from multiple aspects.The results showed that these two vaccines both provided ideal immunoprotection efficiency against E.ictaluri and Y.ruckeri infection in channel catfish,indicating that these two vaccines constructed in this study could effectively enhance channel catfish immunocompetence,and provided better immune defense and protection against E.ictaluri and Y.ruckeri infection.Although the protective effect of bivalent genetic subunit vaccine was slightly better than that of oral vaccine,considering aquaculture production practice,bivalent genetic oral vaccine with orally immunization would be more easily recognized and accepted.