Construction And Evaluation Of The Immunogenicity Of Brucella S2-BCSP31 Deletion Strain For Vaccine

Brucellosis is one of the most important zoonotic diseases caused by Brucella spp.that is of worldwide distribution,especially in developing countries.Brucella spp.,as a typical intracellular pathogen,have a strong invasive ability,and a wide host of infections.More than 60 species of animals are known to be infected with Brucella spp.,Pigs,cattle,and sheep are susceptible animals,and there are cross-infections among livestock.In recent years,with the rebound of the cattle and sheep brucellosis,the sika deer brucellosis in Jilin province has occurred frequently,Diseased deer has become an important source of infection for human,and it seriously affects national economy and has a serious impact on human health and animal husbandry development.Vaccination is the primary measure for brucellosis prevention and control.However,antibody is produced continuously after vaccination,and the routine serological detection method can not distinguish between natural infection and vaccine immunization,which is not conducive to the prevention and control and elimination of brucellosis.BCSP31 gene is a protective antigen of Brucella with good immunogenicity.Some studies have shown that the deletion of it does not affect the survival of Brucella,and it can be the target gene of the deletion vaccine.Therefore,based on the analysis of the epidemic situation and related risk of Brucella in Jilin,we used S2 vaccine strain as parent strain to construct S2-BCSP31 deletion strain vaccine,and evaluate the safety and effectiveness of it.The ELISA method was established with BCSP31 protein as coating antigen,and the feasibility of using the S2-BCSP31 deletion strain as candidate vaccine strain was analyzed.Finally,the S2-BCSP31 infection model of macrophage was established to analyze the mechanism of the BCSP31 gene in Brucella spp.,in order to provide a theoretical basis for the prevention and control of brucellosis and pathogenic mechanisms.The main test results are as follows:1.In this study,458 deer blood samples were surveyed for RBPT and Fluorescence quantitative PCR identification.Atotal of 57 positive samples and 4 suspected samples were tested by RBPT;A total of 59 positive samples were tested by Fluorescence quantitative PCR,and the positive rate was 12.9%.Among them,27 were B.suis,19 were B.melitensis and 13 were B.abortus,Season is the risk factor for Brucella infection.2.The S2-BCSP31 vaccine was constructed by homologous recombination technology.The tests of morphological,growth characteristics and biochemical characteristics proved that the S2-BCSP31 deletion vaccine had the same biological characteristics with S2 vaccine strains.S2-BCSP31 had good genetic stability for 20 generations and 12 months of storage by PCR amplification.Western Blot identification showed that the deleted strain did not react with the hyper-free serum of rBCSP31 protein,indicating that BCSP31 protein was not expressed in the S2-BCSP31 strain,and the S2-BCSP31 strain was constructed successfully.3.In order to explore the role of BCSP31 on the secretion of inflammatory factors and autophagy interaction in macrophages,the model of macrophage infection in vitro of S2-BCSP31 and S2 vaccine strain was established.The results showed that S2-BCSP31 is beneficial to the expression of IL-12 and IL-1 ?,but it inhibits the expression of IL-6 to induced autophagy of macrophages.TNF-?,the LC3 B,Beclin,p62,Bcl2 protein were high expression.The PI3K/Akt/mTOR and MER/ERK pathway of autophagy induced by Brucella was analyzed,which confirmed that the S2-BCSP31 deletion strain could activate autophagy in the PI3K/Akt/mTOR pathway.After inhibiting this pathway,the expression level of autophagy related gene LC3 B,Beclin and ULK mRNA were significantly decreased,and the number of intracellular colonies decreased,indicating that the autophagy of this pathway is beneficial S2-BCSP31 deletion strain survival in macrophages.4.After immunization,the biosafety of the S2-BCSP31 strain was estimatd.The results showed that the spleen weight and spleen CFU in mice injected by S2 and S2-BCSP31 were basally same,there was no significant difference between the two groups.The results of antibody and cytokine detection showed that there was no significant difference in humoral immunity and cellular immunity induced by S2-BCSP31 deletion strain and S2 strain.Mice model of protection test proved that S2-BCSP31 and S2 possess the same protective efficacy against M28,2308 and 1330 after being immunized.Spleen weight and CFU in the immunized group were significantly different from those in the unimmunized group(P < 0.01),but there was no different between S2-BCSP31 and S2,which suggested that the S2-BCSP31 strain can provide the same protective effect as S2 strain,and the S2-BCSP31 vaccine has good potential.5.With rBCSP31 protein as antigen,ELISA detection method was established,by which M28,2308,1330 and S2 sera were detected as positive and S2-BCSP31 as negative.ELISA could be used to discriminate sera from S2-BCSP31 immunized mice and wild-type infected,which provides a theoretical and practical foundation for the establishment of a matching detection method for the labeled vaccine.In summary,the S2-BCSP31 deletion vaccine constructed in this study has good safety and protection.It can distinguish between S2-BCSP31 infection and wild-type infection from the serological perspective,and has a good application prospect.It is of great significance to the prevention,monitoring,elimination and understanding the pathogenesis of brucellosis.