| Salmonella enterica serovar Enteritidis(S.Enteritidis)is poultry high separation rate of Salmonella serotype,via the fecal-oral transmission,after the infection can cause a host of gastroenteritis and sepsis,inflammatory bowel disease disease prevention and control is of great significance to public health.Due to the problem of bacterial resistance and drug residues,antibiotic control is limited,so it is necessary to develop safer and more effective ways to prevent and control S.Enteritidis.The genetically engineered attenuated live vaccine constructed by knockout virulence gene has the advantages of comprehensive immune effect,good safety and low cost,which has a good market prospect.In the process of constructing live attenuated vaccine,the selection of attenuated candidate genes is particularly important,which can effectively reduce the virulence of wild strains without losing their original immunogenicity and can be used as ideal attenuated target genes.Small RNA RyhB-1 has the ability to regulate iron metabolism homeostasis,oxidative stress and other intracellular life activities,and can regulate the expression of type Ⅲ secretorysystem(T3SS)effect protein SopE at the post-transcriptional level,reducing the virility of S.Enteritidis.In this study,the regulatory ryhB-1 gene and virulence invasive gene sopE in S.Enteritidis were used as candidate genes for the construction of attenuated live vaccine.ryhB-1 and sopE were knocked out by λ-Red homologous recombination technology,and the gene deletion strain 50336ΔryhB-1ΔsopE of S.Enteritidis was successfully constructed.The biological characteristics,in vitro and in vivo safety and immunogenicity of the deletion strain were determined.Meanwhile,the immune protection effect of the deletion strain on chicks was further explored by simulating natural infection with oral challenge,and the feasibility of using the deletion strain as a live attenuated vaccine for genetic engineering was preliminatively investigated.1.Construction of recombinant S.Enteritidis strain 50336ΔryhB-1ΔsopEThe homologous arm primer was designed according to the sopE gene sequence of S.Enteritis published by GenBank.Using pKD3 plasmid as the template,PCR amplified the gene fragment containing chloramphicol resistance,the fragment was electrically converted into 50336ΔryhB-1 receptor cells which containing pKD46 plasmid.A single homologous recombinant strain 50336ΔryhB-1ΔsopE::cat was obtained by PCR.The temperature sensitive plasmid pCP20 was electrically transferred into the primary recombinant bacteria,the chloramphenicol resistance gene was eliminated,and the recombinant bacteria was identified by PCR.The 50336ΔryhB-1ΔsopE deletion strain of S.Enteritis was obtained.2.Preliminary evaluation of the potential of recombinant S.Enteritidis strain 50336ΔryhB-1ΔysopE as a live attenuated vaccine candidateIn order to evaluate the safety of S.Enteritis 50336ΔryhB-1ΔsopE deletion strain,LD50 of the deletion strain against 1-day-old Rugao yellow chickens was first determined.The results showed that the virulence of the deletion strain was significantly reduced compared with wild strains and supplemented strains.The results of bacterial adhesion and invasion to intestinal epithelial cell Caco-2 showed that the double deletion of ryhB-1 and sopE genes resulted in a significant decrease in adhesion and invasion compared with the wild strain and the replacement strain(p<0.05),indicating that the deletion strain had lower colonization and invasion ability to host cells and weakened virulence in vitro.After oral inoculation of 3-day-old Rugao yellow chickens with the minimum inoculation dose,the results showed that the deletion strain had lower colonization ability in chicks than the wild strain,and the deletion strain could still be isolated in the organs on the 14th day after inoculation,which would not be quickly cleared by the body.These results indicate that the absence of ryhB-1 and sopE can lead to the weakening of the virulence of S.Enteritidis in vivo and in vitro,which also has good safety.To evaluate the immunogenicity of the deletion strain,1-day-old Rugao yellow chickens were orally inoculated with 1×109 CFU/mL,which inoculated with the same dose and inoculation method two weeks later.The results showed that serum IgG antibody could be detected in the immune group after two weeks of immunity,and the antibody levels of the wild strain,the deletion strain and the commercial vaccine strain were higher than those of the blank group during the experiment,which could induce good humoral immunity.The intestinal mucosal IgA antibody of chicks in all immune groups remained at a low level at the initial stage of immunization,which began to increase significantly after the second immunization.At this time,the antibody level in all immune groups was significantly higher than that in the blank group,inducing an efficient intestinal mucosal immune response.After immunization,the expression levels of various cytokines in peripheral blood lymphocytes of chicks were detected by fluorescence quantitative PCR.After immunization,the expression levels of IL-6,IL-10,IFN-γ and TNF-α were significantly up-regulated in all immune groups,resulting in strong Thl and Th2 immune responses.There was no significant difference of the antibody levels among the deletion strain,the wild and commercial vaccine strains,indicating that the deletion strain could induce efficient and durable humoral,mucosal and cellular immunity while decreasing virulence,also had good immunogenicity.In order to evaluate the protective effect of the deletion strain on chickens,the chickens were artificially infected with S.Enteritidis wild strain at high dose after immunization.The results showed that the oral immunization of the deletion strain and the commercial vaccine strain provided 70%and 80%protection rates for chickens.The immunization of the deletion strain could effectively reduce the morbidity of chickens infected with S.Enteritidis wild strain,also alleviate the damage of organs and tissues caused by pathogenic bacteria infection.The above results showed that the knockout of ryhB-1 and sopE genes of S.Enteritidis weakened the virulence of the wild strain while still retaining its original immunogenicity,which not only had stable safety but also effectively stimulated the overall immune response of the host,providing a good protective effect against the challenge of high-dose wild strains of S.Enteritidis.This study provides experimental basis for the development of live attenuated vaccine against S.Enteritidis. |
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