81 / 2024-10-25 18:56:43

Construction of a Multi-Epitope Subunit Vaccine Against Salmonella Enterica Serovar Enteritidis and Analysis of Its Immunoprotective Efficacy

Salmonella enterica subsp. enterica serovar Enteritidis (SE) is a facultative intracellular pathogen, which can be carried asymptomatic in the intestines or gallbladder of livestock and poultry, and can cause gastroenteritis, which is one of the main causes of food-borne diseases in the world. Antibiotic therapy is the main means to fight against Salmonella Enteritidis infection. However, in view of the increasing number of multi-drug-resistant Salmonella in animals and the harm of drug residues, the development of safe and effective Salmonella vaccine is becoming more and more important. Common inactivated vaccines can not cause good cellular immunity, and attenuated live vaccines are at risk of returning to strong virulence. Multi-epitope subunit vaccine can induce a strong specific immune response by using the least immunogenic components, and can avoid the possibility of the virulence of attenuated live vaccine returning to strength. At present, the study of subunit vaccine with the dominant epitopes of Salmonella B cells and T cells in series mainly stays in the stage of computer design and prediction, but no animal experiment has been carried out to evaluate the immune protection efficiency. The purpose of this study is to construct a multi-epitope subunit vaccine of flagellin and secretory system type Ⅲ (T3SS) system protein SipD of Salmonella Enteritidis by bioinformatics software, and to explore its preventive and protective effects on Salmonella Enteritidis through animal experiments. The main contents are as follows:
1. Design of multi-epitope subunit vaccine FLPD for Salmonella Enteritidis and prediction of immune effect. FliC protein and SipD protein were selected as antigen proteins, and the amino acid sequences of the two proteins were obtained on National Center for Biotechnology Information (NCBI). 10 dominant B cell epitopes and 3 dominant T cell epitopes were obtained by online analysis software ABCpred and EpiJen. The epitope protein FLPD was obtained by connecting all the dominant B and T cell epitopes in series with “GSGGSG” and “AYY”, respectively. FLPD protein is stable, with a hydrophilic index of -0.704 and a good hydrophilic index. It has no transmembrane structure and is extracellular, and there is no signal peptide signal, which is beneficial to protein expression. The results of electronic immune simulation showed that the expression of interferon in specific cytokines increased, and both B cell population and T cell population increased.
2. Construction, expression, purification and verification of multi-epitope subunit vaccine FLPD against Salmonella Enteritidis. The recombinant prokaryotic expression plasmid pET-28b(+)-FLPD containing the target gene FLPD was constructed and transformed into competent cells of E. coli BL21(DE3) to obtain recombinant expression bacteria. The expression of FLPD protein with His tag was induced by IPTG inducer. The results of SDS-PAGE and Western blot showed that FLPD successfully expressed a soluble antigen with a size of 35.0 kDa at IPTG concentration of 1.0 mmol/L, temperature of 37℃ and induction time of 4 h. The protein was purified by His tag purification column. The purification results showed that the purity of the target protein was high when the concentration of imidazole was 250 mmol/L, and the purity of the protein was 95.6%.
3. Evaluation of immune protection effect of multi-epitope subunit vaccine FLPD against Salmonella Enteritidis in mice. In this study, 6-week-old female BALB/c mice were selected as the research object, and PBS group, 20 µg protein group, 50 µg protein group and 90 µg protein group were set up, and Freund's adjuvant was selected. After each immunization, the specific antibody IgG level in serum was detected, which showed that the specific antibody IgG level increased significantly after immunization (P<0.001). The expressions of cytokines IFN-γ, IL-4, IL-6 and IL-12 in vivo were significantly up-regulated (P<0.01), indicating that protein FLPD can induce both cellular immunity and humoral immunity. After immunization, the living conditions of mice were normal and the vaccine was safe. The Median Lethal Dose (LD50) of Salmonella enterica subsp. enterica serovar Typhimurium (ST) CVCC541 to female BALB/c mice was 1.20×104 Colony Forming Unit (CFU) and 6.03×102 CFU, respectively. After immunization, BALB/c mice were challenged with 10LD50 of Salmonella Enteritidis CMCC(B)50335 (1.20×105 CFU) and Salmonella Typhimurium CMCC541 (6.03×103 CFU), and the survival rates of immunized mice were 65% and 45% respectively. All the mice in the non-immunized group died, and the bacterial load of the liver and spleen in the immunized group was significantly lower than that in the control group (P<0.001), and the pathological changes of intestinal tissue in the immunized group were lower than that in the control group. This proved that the multi-epitope vaccine FLPD had a certain protective effect on mice infected with Salmonella Typhimurium.
4. Evaluation of Immune Effect of multi-epitope Subunit Vaccine FLPD against Salmonella Enteritidis on Chicks. Salmonella Enteritidis CMCC(B)50335 was used to challenge the Four weeks old White Leghorn chicks by intramuscular injection, and the calculated LD50 was 6.31×1010 CFU. Chicks were immunized with PBS, commercial live attenuated vaccine (Sm24/Rif12/Ssq strain) and FLPD vaccine respectively. The 10LD50 of Salmonella Enteritidis (6.31×1011 CFU) was used to attack chicks. The results showed that the protection rate of commercial attenuated live vaccine was 80% and that of FLPD vaccine was 70%.
To sum up, this study is the first time to construct a multi-epitope subunit vaccine for FliC of Salmonella Enteritidis and SipD. The prepared multi-epitope subunit vaccine FLPD has a good protective effect on Salmonella Enteritidis infection in mice and chicks, and has a cross-protective effect on Salmonella Typhimurium. It lays a foundation for the subsequent development of safer and more effective multi-epitope subunit vaccine of Salmonella Enteritidis.