Construction Of Recombinant Bacillus Subtilis Expressing Salmonella Enteritidis Multi-epitope Protein And Evaluation Of Its Immune Effect On SPF Chickens

Salmonella is one of the most important pathogenic bacteria that cause food-borne diseases.It can infect pigs,chickens and other edible animals and pass it to humans through the food chain,thus causing harm to public health.There are more than 3,000 serotypes of Salmonella,among which Salmonella Enteritidis(S.Enteritidis)showed the highest isolation rate.Poultry is one of the most important carriers of S.Enteritidis.Once Salmonella infection occurs,it will cause serious economic losses.Antibiotics and vaccination are the most important means of prevention and control of Salmonella.In recent years,the use of antibiotics has been limited because the abuse of antibiotics has led to the very serious bacterial resistance.Currently,the main Salmonella vaccines on the market are inactivated or attenuated vaccines,but both two kinds of vaccines have certain limitations.Inactivated vaccines can't induce cellular and mucosal immunity,and attenuated vaccines are easy to cause infections in chickens with low immunity.Therefore,it is urgent to develop a vaccine with good immune effect,high safety and few side effects.Live-vector vaccines of probiotics have been widely used against microbial infections in animals and can offer an excellent safety profile.As a food-grade probiotic,Bacillus subtilis itself has the advantages of regulating intestinal flora,improving animal growth performance,enhancing immunity,and short fermentation period.It is considered as a good live vaccine carrier,and has made a significant progress in the prevention of viral,bacterial and parasitic diseases.In order to improve the immunogenicity of the vaccine,we selected 4 antigenic proteins(Omp C,Fli C,Sop F,Sse B),their secondary structure,hydrophilicity,flexible region,surface accessibility and antigen index were predicted with DNA Star software,and B cell and T cell epitopes were analyzed and predicted with the online website IEDB,SYFPEITHI and Net MHCpan-4.1,then analyzed and compared their prediction results,concatenate the regions where T cell epitopes are concentrated,and CHIL-18 was added to the tail as an immune adjuvant.We cloned the synthesized gene sequence into the Bacillus subtilis-Escherichia coli shuttle vector p HT43,and then transformed the recombinant expression plasmid into the Bacillus subtilis by electroporation.Western-Blot identifies whether the target protein is successfully expressed.The result shows that there is a clear band near 64kda,indicating that the target protein is successfully expressed.To evaluate the immune effect of recombinant Bacillus subtilis,we further performed multiple immunoassays.Eighty 1-day-old SPF chicks were randomly divided into 4 groups and were orally immunized with the same dose of PBS,empty Bacillus subtilis WB800N-p HT43,recombinant Bacillus subtilis WB800N-p HT43-HA,and the commercially available attenuated vaccine Avi Pro Salmonella.vac E,first immunization on days 1-3,1 m L(1×10¹⁰CFU)of recombinant Bacillus subtilis(PBS)was administered daily,and second immunization on days 14-16.14 days after the second immunization,peripheral blood,jejunal lavage fluid,tracheal lavage fluid,and spleen(6 animals in each group)were collected,the serum-specific antibody Ig G and mucosa-specific antibody s Ig A levels were detected by indirect ELISA.The serum cytokines IL-2 and IL-6 were detected by ELISA,and the levels of CD4⁺and CD8⁺in peripheral blood and spleen were detected by flow cytometry.At the same time,10 chicks were randomly selected from each group and injected intraperitoneally with 1×LD₅₀of S.Enteritidis.The death of each group was counted for 7 consecutive days,and the histopathological examination of the intestine and spleen was performed one week later.The results showed that 14 days after the second immunization,the specific s Ig A levels in the jejunal lavage fluid and tracheal lavage fluid of the chickens in the recombinant Bacillus subtilis group were significantly higher than those in the other groups.The specific Ig G level was also significantly increased,indicating that recombinant Bacillus subtilis could effectively induce mucosal immunity and systemic immunity.The results of flow cytometry showed that the proportion of CD4⁺and CD8⁺T lymphocytes in the spleen and peripheral blood of chickens in the recombinant Bacillus subtilis group was significantly increased;the results of the spleen lymphocyte proliferation experiment showed that the recombinant Bacillus subtilis could significantly stimulate the proliferation of spleen lymphocytes.The results indicated that recombinant Bacillus subtilis could effectively induce cellular immunity.In terms of cytokines,the level of serum IL-2 in the recombinant Bacillus subtilis group was significantly increased,and the level of IL-6 was significantly decreased.Post-challenge spleen,jejunum pathological analysis and mortality statistics showed that both recombinant Bacillus subtilis and the attenuated vaccine could reduce the pathological damage of spleen and jejunum caused by S.Enteritidis infection,and reduce the mortality of chicks.In this study,by predicting the epitopes of the S.enteritidis proteins Omp C,Fli C,Sop F and Sse B,the Bacillus subtilis that can express the Omp C-Fli C-Sop F-Sse B-CHIL-18 fusion protein was constructed.Studies have shown that the recombinant Bacillus subtilis we constructed has a good immune protection effect on SPF chicks,and can effectively induce humoral immunity and cellular immunity.This study provides theoretical data for the study of live vector vaccines of S.Enteritidis and technical reference for the study of recombinant Bacillus subtilis vaccines.