Preparation Of Riemerella Anatipestis Subunit Vaccine And Evaluation Of Its Immune Effect

This study for Guizhou main epidemic serotype 1,2 bivalent inactivated vaccine,and prepare RA Omp A genetic engineering subunit vaccine immunization duck respectively,by establishing the ELISA method detection serum RA specific antibodies and cytokine content and histopathological analysis,compare bivalent inactivated vaccine and genetic engineering vaccine subunit vaccine induced duck immune response,in order to provide an effective way for the prevention and control of RA disease in Guizhou.The 16Sr RNA and RA-OMPA genes of 18 RA strains（RA-S01～S16,RA-HS01,RA-G06）were analyzed by homology and evolutionary tree.The results showed that the nucleotide homology of 18 RA 16Sr RNA genes was 96.3%-99.2%.The homology of RA-OMPA gene was 92.6～99.7%;the bioinformatics software analysis and prediction results showed that RA-Omp A protein has more dominant antigen epitope structure and can be applied to immunological studies.Primers were designed based on the Omp A gene sequence in RA（G06 strain）and the cloning sites in the p ET32a prokaryotic expression vector sequence,The Omp A gene was amplified and inserted into the p ET32a prokaryotic expression vector,To construct the recombinant prokaryotic expression plasmid p ET32a-Omp A,E.coli BL21（DE3）competent cells were transformed after identification by plasmid PCR,double digestion and sequencing,After the induction,sonication,and purification of the recombinant bacteria,The Omp A recombinant protein size was identified as about 57 KD by SDS-PAGE and Western blotting analysis;The ELISA method was initially established and optimized for reaction conditions by using purified Omp A recombinant protein as coated antigen,After multiple square array detection results show;The Omp A protein was coated at a concentration of 1μg/m L,Serum was diluted at 1:200,The packets were conditioned for 37℃incubation for 2 h after another 4℃overnight,In 5%BSA,The closure condition was 37℃,30min,The serum reaction time was 37℃for 1 h.The dilution of the enzyme-conjugated secondary antibody was 1:400.The reaction time of the enzyme-conjugated secondary antibody was 37℃for 2 h.Display liquid reaction time is 15 min.critical value OD₄₅₀For the value of 0.0813.The indirect ELISA method established in this study showed strong specificity,high sensitivity and good repetition.A subunit vaccine with a final concentration of 500μg/m L was prepared with Capom as an adjuvant.At the same time,serum type 1 RA（G06 strain）and serum type 2 RA（HS01 strain）were selected to prepare the bivalent inactivated vaccine.Through the quality test of the inactivated vaccine,its safety and stability were good.The 7-day-old 120 healthy chicks were randomly divided into 30 mice with commercial bivalent inactivated vaccine（RAf63+2 RAf34）,0.5 m L,self-developed bivalent inactivated vaccine（RA（G06）+2 RA（HS01））,0.5 m L of recombinant Omp A subunit vaccine 500 g and 0.5 m L of saline.Reimmunization one week apart（i.e.,14 days of age）.The peripheral blood was collected from 10 w ducks before immunization（0 w）and 1 w（7 days old）,1 w（14 days old）,3 w（21 days old）,4 w（28 days old）,6 w（42 days old）and 7 w（49 days old）.OD as determined according to the established Omp A indirect ELISA assay₄₅₀Values,serum Th1（IL-2,IFN-α）,Th2（IL-4）and tumor necrosis factor（TNF-α）were determined by ELISA.At the same time,RA serum type 1（G06）and HS01)and RA serum type 2（HS01）for 7 d,twice the lethal dose,isolated feeding for 7 d,and pathological analysis of heart,liver,spleen and brain.The results showed that OD measured by Omp A indirect ELISA by O m p A₄₅₀Value,the subunit vaccine group and the commercial bivalent inactivated vaccine group were higher than the self-developed bivalent inactivated vaccine group;but the serum IL-2,IFN-,IL-4 and TNF-commercial bivalent inactivated vaccine group and the self-developed bivalent inactivated vaccine group were higher than the subunit vaccine group.The results of challenge protection test showed that the protection rate of serum type 1（G06 strain）,serum type 2（HS01 strain）（90%,80%）and self-developed bivalent inactivated vaccine（90%,90%）was higher than that of the subunit vaccine group（40%,30%）.It was revealed that there were few cellulose exudates,large hepatocyte swelling,necrosis,large spleen bleeding,unclear red and white pulp,satellite brain and swelling of nerve cells.The commercial bivalent inactivated vaccine group and the self-developed bivalent inactivated vaccine group had mild pathological changes compared with the normal saline group.Subunit vaccine can protect against RA serotype 1 and type 2,but the protection is not as good as the bivalent inactivated vaccine.The reason may be that the bacterial genome is so large that the use of a relatively single antigen component cannot provide effective protection,and only multiple synergistic actions of proteins or other antigenic components can provide effective protection.Duck immune responses induced by bivalent inactivated vaccines and genetically engineered subunit vaccines were compared by an established ELISA test in serum for RA-specific antibodies and cytokines content and histopathological analysis.This trial provides a reference for the prevention and control of RA disease by vaccines.