Development Of Vaccines Derived From A Guizhou Epidemic Strain Of Riemerella Anatipestifer And Their Immune Efficacy

Riemerella anatipestifer infection（RAI）is caused by Riemerella anatipestifer（RA）infection of ducks,geese,turkeys and other poultry.Acute,contact,and septic infectious diseases characterized by inflammation,air sacculitis and meningitis.The disease has a high morbidity and mortality rate and is widespread all over the world.It is one of the main bacterial infectious diseases that seriously harm the duck industry.There are many serotypes of RA,and there is a lack of cross-immunity protection between different serotypes,and RA is prone to resistance to antibacterial drugs,which brings difficulties to the prevention and control of RAI and seriously affects food safety.Therefore,it is more urgent to develop a new RA vaccine.RA outer membrane protein A（OmpA）and co-hemolysin（CAMP）are highly conserved in different serotypes of RA strains and have good immunogenicity.They can be used as candidate proteins for the development of genetic engineering subunit vaccines.Therefore,based on the cloning and analysis of the OmpA and CAMP genes of the RA Guizhou isolate,the OmpA-CAMP fusion gene was constructed,and the r OmpA-CAMP fusion protein was obtained as a subunit vaccine through the prokaryotic expression system.At the same time,the RA Guizhou epidemic strain propolis inactivated vaccine was prepared to immunize ducklings,and the immune response induced by the r OmpA-CAMP fusion protein and propolis inactivated vaccine was evaluated by detecting the relevant immune indicators of the ducklings,it is expected to lay the foundation for the development and application of new RA vaccines.1.Serotype identification of RA Guizhou isolates and cloning and analysis of OmpA and CAMP genesIn order to clarify the serotype of RA Guizhou isolates and their OmpA and CAMP gene characteristics,this experiment was conducted on 13 RA Guizhou isolates（RA-SS-1～12 strains and RA-AS-1 strains）using 16S rRNA gene-specific primers.PCR detection and analysis,use different serotypes of RA positive sera for slide agglutination test,and then respectively use RA OmpA and CAMP gene specific primers for PCR amplification,the PCR products were recovered for cloning and bioinformatics analysis,the results showed:13 RA strains all the isolates from Guizhou could amplify specific fragments of the 16S rRNA gene.Sequencing revealed that the nucleotide homology of the specific fragments among the 13 strains was98.5%～99.9%,which was the same as that of the 15 reference strains on Gen Bank,and their origin is 97.5%～99.9%;serotype identification shows that RA-SS-1～10 strains are serotype 2RA,RA-SS-11～12 strains are serotype 11 RA,and RA-AS-1 strains are Serotype 3 and 11positive sera have cross-agglutination reactions.Cloning analysis showed that the full length of OmpA gene of 13 RA Guizhou isolates was 1 164 bp,which could encode 387 amino acids.The nucleotide homology between the strains was 100%,and the nucleotide homology to the reference strain RA Yb2 Up to 100%;the CAMP gene of 13 RA Guizhou isolates is 1 026 bp in length,which encode 341 amino acids.Except for the synonymous mutation at base 624 of RA-SS-7 strain,the remaining 12 nucleotides the homology is 100%,and the nucleotide homology to the RA standard strain ATCC-11845 is 100%;RA Opm A and CAMP proteins are hydrophilic proteins,without transmembrane and signal peptide regions,and contain more advantages Epitope structure.These results indicate that the dominant serotype of the 13 RA Guizhou isolates is serotype 2,and the OmpA and CAMP genes are highly conserved and have good antigenic characteristics,which can be used as candidate proteins for the preparation of RA genetically engineered subunit vaccines.2.Preparation of OmpA,CAMP and OmpA-CAMP subunit proteins of the RA Guizhou epidemic strainRandomly selected the RA Guizhou epidemic strain（RA-SS-8 strain）OmpA and CAMP gene plasmid as template,design and synthesize specific primers with linker-（G₄S）₃,use SOE-PCR technology to amplify OmpA-CAMP fusion gene,and combine OmpA,CAMP and OmpA-CAMP genes were cloned into pET-28a（+）vector,recombinant expression plasmids were constructed,and Escherichia coli Rosetta（DE3）was transformed to induce IPTG expression.After inducing expression conditions were optimized,the expressed protein was identified by SDS-PAGE and Western blot.The results showed that:pET-28a-OmpA,pET-28a-CAMP and pET-28a-OmpA-CAMP were successfully constructed.The molecular weights of rOmpA,rCAMP and rOmpA-CAMP fusion proteins induced by IPTG were approximately 45 k Da,37k Da and 83 k Da,respectively.Among them,r CAMP protein is expressed in soluble form,and r OmpA and r OmpA-CAMP proteins are expressed in the form of inclusion bodies;the optimal inducible expression conditions for r OmpA,r CAMP and r OmpA-CAMP fusion protein are 37℃0.2 mmol/L IPTG induced expression for 5 h,respectively,18℃0.4 mmol/L IPTG induced expression for 12 hours and 37℃0.2 mmol/L IPTG induced expression for 5 hours;r OmpA and r CAMP protein can be purified by Ni-NTA affinity chromatography,r OmpA-CAMP protein should be purified by gel-cutting purification method;Western blot showed that purified r OmpA,r CAMP and r OmpA-CAMP proteins can react specifically with His-tag monoclonal antibodies.These results indicate that this experiment successfully expressed and purified r OmpA,r CAMP and r OmpA-CAMP proteins.3.Preparation of inactivated propolis vaccine against RA Guizhou epidemic strainRandomly selected the RA Guizhou epidemic strain（RA-SS-8 strain）was used as the basic strain.After growth curve determination and inactivation condition screening,the RA Guizhou epidemic strain propolis inactivated vaccine was prepared with propolis as an adjuvant,and it was sterile and safe.The test results showed that when the OD₆₀₀ value of the RA-SS-8 strain was 0.1-0.8,the strain was in the logarithmic growth phase,and the OD₆₀₀ value and the number of viable bacteria showed a good linear relationship;the RA-SS-8 strain was best inactivated The conditions were 0.2%formaldehyde solution and inactivated at 37°C for 12 h;the inactivated propolis vaccine contains 3.8×10⁹ CFU/mL,and the dry matter content of propolis was 10 mg/mL;sterility test showed that it was not seen on the chocolate agar medium Colony growth;safety inspection showed that ducklings inoculated with 2 times the immunization dose showed no adverse reactions during the observation period,and no obvious lesions were observed in gross lesions.These results indicate that this experiment successfully prepared an inactivated propolis vaccine against the RA Guizhou epidemic strain.4.Immune effect analysis of RA Guizhou epidemic strain subunit vaccine and propolis inactivated vaccineA total of 210 healthy ducks aged 7 days were selected and randomly divided into fusion protein group,fusion protein+CpG ODN group,combined immunization group,propolis vaccine group,commercial vaccine group and PBS control group,each with 35 ducks,corresponding to r OmpA-CAMP Fusion protein,r OmpA-CAMP fusion protein+CpG ODN,r OmpA+r CAMP recombinant protein,propolis inactivated vaccine,bivalent inactivated vaccine and sterile PBS solution immunization（first immunization at 7 days,second immunization at 21 days）,before the first immunization（0 d）and 7 d,14 d,21 d and 28 d after immunization,randomly collect the peripheral blood samples and spleen tissues of the test ducks,5 per group per time period,and use the ELISA method to detect duck serum RA antibody,CCK-8 method to detect the proliferation of peripheral blood lymphocytes in ducks,q RT-PCR method and detection of duck spleen IL-2,IL-4,IL-6,IFN-αand IFN-γgene transcription levels,ELISA method to detect ducks Serum IFN-γand IL-4 secretion levels,the results show that:subunit vaccine and propolis inactivated vaccine can induce ducks to produce RA-specific antibodies and stimulate the proliferation of duck peripheral blood lymphocytes,and induce duck spleen IL-2 and IL-4,IL-6,IFN-αand IFN-γgene transcription levels are up-regulated,which promotes the increase of IFN-γand IL-4 secretion levels in duck serum.The fusion protein+CpG ODN group has a significant effect in the subunit vaccine（P﹤0.05）.These results indicate that both subunit vaccines and inactivated propolis vaccines can induce good immune responses in ducks.5.Immune protection analysis of RA Guizhou epidemic strain subunit vaccine and propolis inactivated vaccineIn the fusion protein group,the fusion protein+CpG ODN group,the combined immunization group,the propolis vaccine group,the commercial vaccine group and the PBS control group,the newly prepared strain of RA-SS-8 was injected intramuscularly on the 14th day（28 days after the first epidemic）after the second immunization,10 duck per group,2×10⁸ CFU/0.5 m L per duck,the immune protection rate was observed and calculated.At the 7th day after challenge（the 35th day after immunization）,the heart and liver tissues of the experimental ducks were collected and pathological sections were made to observe the histomathological changes.The results show that:the fusion protein group,fusion protein+CpG ODN group,combined immunization group,propolis vaccine group,commercial vaccine group and PBS control group have 20%challenge protection rates,30%,20%,70%,100%and0%.On the 7th day after the challenge,the outer membrane of the heart tissue in the PBS control group was thickened with a large amount of fibrinous exudate on the surface,and the liver tissue was thickened with a large amount of pink reticular cellulose on the surface,and the liver cord was arranged disorderly;The fusion protein group and the combined immunization group showed slight pathological changes compared with the control group;the fusion protein+CpG ODN group,propolis vaccine group and commercial vaccine group had no obvious pathological changes.These results indicate that the RA subunit vaccine can induce immune protection in ducks,but it cannot resist the RA Guizhou epidemic strain（RA-SS-8 strain）as effectively as the propolis vaccine and commercial vaccines.In summary,the dominant serotype of RA Guizhou isolate is serotype 2.The OmpA and CAMP genes of RA Guizhou isolate are highly conserved,and the overall antigenicity of the encoded protein is good;the constructed recombinant plasmids can be well obtained in E.coli expressed,and can induce ducks to produce good humoral and cellular immune responses,but can not effectively resist the attack of RA Guizhou epidemic strain（RA-SS-8 strain）;the prepared RA Guizhou epidemic strain propolis inactivated vaccine can induce Duck produces good humoral immunity and cellular immune response,and has a good protective effect.These research results provide a scientific basis for the development of a new RA vaccine.