The Immune Protection Of Recombinant Attenuated S. Typhimurium O Antigen Against O₁,O₂ And O₇₈ Of Avian Pathogenic Escherichia Coli

Avian pathogenic Escherichia coli?APEC?serogroup of O₁,O₂ and O₇₈ are the most frequently isolated serogroups from field infections.It has been known that specific immune response against O antigen polysaccharide can provide protection against APEC infection.As there are currently no effective vaccines derived from O antigen polysaccharide available,the main purpose of this paper is remodling Salmonella O antigen polysaccharide to provide protection against prevalence serogroup of APEC O₁,O₂ and O₇₈.The main contents and results are described below.1.Shuttle plasmids construction to assemble O antigen polysaccharideThe PCR amplified fragments of ARS4/CEN5-TRP-Spec with yeast origin,selection marker and ori-asd-T1T2 with prokaryotic origin,selection marker were recombinated by Gibson assembly method,yielding to four yeast-prokaryote shuttle plasmids pSS25,pSS26,pG8R210 and pG8R211.2.APEC O₁,O₂ and O₇₈ gene cluster assemblyYeast transformation mediated recombination?TAR?was adopted to assemble APEC O₁,O₂ and O₇₈ O antigen,gene cluster of 10.9 kb,15.9 kb,13.2 kb,26.9 kb,29.1 kb and 40.1 kb were assembled into shuttle plasmids pSS26 to obtain recombinant plasmids pSS27,pSS28,pG8R206,pG8R207,pG8R208 and pG8R209.3.Construction of Salmonella mutant strains to deliver APEC O antigenSuicide plasmids were used to delete asd,crp,cya and rfbP from S.Typhimurium to construct mutant strains of S184??asd-66?,S185??asd-66?rfbP45?,S739??asd-66?crp-24?cya-25?and S740??asd-66?crp-24?cya-25?rfbP45?for O antigen delivering.4.Biological characteristics analysis of recombinant S.Typhimurium expressing APEC O antigenPlasmids of pSS27,pSS28 and pG8R206 were electrotransformed into S740.All of the results detected by serum agglutination,silver stain,western blot and cystal violet staining as well as heat agglutination showed that APEC O antigens were expressed successfully in S740.Stability detection showed that plasmids in S740 andc12441 were kept as much as 85%throughout 5 passages over 3 days culture.Biological characteristics showed that APEC O antigen expression did not influence the growth,attachment,invasion,polymyxin B and DOC sensitivity of S740,while leading to 100-flod decrease in virulence of S184??asd-66?and S185??asd-66?rfbP45?.5.Immunogenicity study of attenuated Salmonella expressing APEC O₁,O₂ and O₇₈Oral and intramuscular immunization,air sac and intramuscular challenge route were selected to analyze immune protection efficacy of S739?pSS27?and S740?pSS27?in Lohmann hens,the results showed that S740 was superior to S739 in delivering APEC O₁ to elicit immune protection,intramuascular immunization elicited better humoral immune and weaker mucosal immune,and vice versa.Oral immunization of S740?pSS27?provided 66.7%protection against APEC O₁ challenge by air sac.Oral immunization of S740?pSS28?afforded71.4%protection against air sac challenge of APEC O₂.Oral immunization ofc12441?pG8R206?against homologous APEC O₇₈ and heterologous O₁ and O₂ challenge by air sac is 95%,10%and 40%,respectively.6.Co-immunization of recombinant strains expressing APEC O₁ and O₂The heterologous immunization of oral prime-intramuscular boost was adopted in the co-immunization of S740?pSS27?and S740?pSS28?,which elicited immune responses as well as protection against both APEC O₁ and O₂,the protection efficacy is 66.67%and73.33%,respectively.7.The impact of O antigen length on biological characteristics and immunogenicity of Salmonella vectorSalmonella with different O antigen length from short,medium to long were constructed by complementing wzz from heterologous strains,animal immune protection assay showed that Salmonella with 15-21 O units possed highest immunogenicity and induced highest immune response.In conclusion,protection against APEC O₁,O₂ and O₇₈ can be achieved by constructing O antigen recombinant S.Typhimurium and the immunogenicity of Salmonella delivery vector can be optimized by altering O antigen length.