Immune Enhancement Effect Of PUC18-CpG Recombinant Plasmid On Riemerella Anatipestifer Inactivated Vaccine

Riemerella anatipestifer（RA）is an important pathogen that causes losses in the poultry industry,and can cause serositis and sepsis in ducks and other birds.The non-standard use of antibacterial drugs has led to a large number of RA strains showing strong drug resistance.Vaccine prevention of RA infection is an important means at present.However,the vaccine has problems such as weak response and low immunogenicity.It has been confirmed that unmethylated oligodeoxynucleotide（Cytosine-phosphate-guanine oligodeoxynucleotide,CpG ODN）can enhance the immunogenicity of various vaccines.However,the production conditions of CpG ODN are harsh and the synthesis cost is high,so it is difficult to apply in actual production.Studies have shown that it is feasible to insert CpG motifs into the p UC-18 vector to enhance the immunogenicity of vaccines.In this study,we screened different structural types of CpG ODNs,constructed a recombinant plasmid pUC18-CpG,and evaluated its enhanced RA immunogenicity in ducks.We designed and synthesized 6 CpG ODN sequences,and showed that the stimulation index of CpG-2,CpG-3,and CpG-6 in duck peripheral blood lymphoid proliferation experiment was highest.The recombinant plasmid pUC18-CpG was constructed,and its immunogenicity was compared with that of CpG ODN.The results showed that the stimulation index of pUC18-CpG recombinant plasmid containing CpG-3 and the relative expression of related cytokines were no significant difference between them（p>0.05）.The above results show that the recombinant plasmid pUC18-CpG can replace CpG ODN as an adjuvant.Using inactivated RA GH5 as antigen and pUC18-CpG recombinant plasmid as adjuvant,ducklings were subcutaneously immunized twice,and antibody titers and cytokine levels were detected.The results showed that compared with RA vaccine,the pUC18-CpG adjuvant group significantly increased serum antibody titers and IL-2 and IL-4 cytokine levels.14 days after the booster immunization,the test ducks were challenged by intramuscular injection of 10 LD₅₀ RA GH5.The results showed that all the ducks in the control group died,the survival rate of RA vaccine was 50%,the survival rate of 80μg pUC18-CpG was 70%,and the survival rate of 40μg and 20μg was 60%.In addition,all pUC18-CpG adjuvant groups improved vaccine immunogenicity and reduced histopathological damage and bacterial load.Therefore,pUC18-CpG adjuvant combined with RA GH5 inactivated vaccine can induce stronger humoral and cellular immune responses.