Development And Evaluation Of Salmonella Enteritidis Subunit Vaccine Based On Manganese Adjuvant

Salmonellosis is an important zoonotic bacterial disease that can cause diarrhea,miscarriage,and sepsis in livestock and poultry,causing huge economic losses of China's breeding industry;at the same time,the polluted livestock and poultry products are one of the main sources with human Salmonella contamination,which seriously affects the safety of animal-derived food.This is a major public health problem.Vaccination is a vital measure currently used to reduce Salmonella contamination.Subunit vaccines have the advantages of less side effects and strong safety;and with the help of adjuvants,they can stimulate more comprehensive immunity response.The SseB protein is a sophisticated protein of the Salmonella type ? secretion system,which has strong immunogenicity,and is highly conserved among various Salmonella serotype.In this study,the proteins rHis-SseB and rGST-SseB were expressed by the E.coli expression system.Then,rHis-SseB protein was set as the immunogen,combined with Manganese adjuvant to conduct immune characteristics analysis and immune effect evaluation through mouse experiments.Later setting rHis-SseB protein and flagellin as immunogen,construct multi-component subunit vaccine combined with Manganese adjuvant.Conduct immune characteristics analysis and immunization effect evaluation based on mouse experiments.The ultimate goal is to provide a new subunit vaccine candidate stock for the national livestock and poultry farming to response to the "Reduce Antibiotic,Replace Antibiotic" campaign,in order to improve the extent of Salmonella control.1 Expression and identification of Salmonella enteritidis recombinant SseB proteinUsing the whole genome of C50041 as a template,the sseB gene was cloned;the recombinant prokaryotic expression plasmids pCold-sseB and pGEX-6P-1-sseB were constructed,and were identified by PCR and enzyme digestion.The recombinant prokaryotic expression plasmid was successfully constructed by sequencing analysis.Recombinant plasmids identified correctly were transformed into competent cells to construct recombinant expression bacteria BL21(DE3)(pCold-sseB)and BL21(DE3)(pGEX-6P-1-sseB).Expression of recombinant proteins rHis-SseB and rGST-SseB were induced by E.coli expression system.SDS-PAGE and Western blot (anti-His and anti-GST monoclonal antibodies as primary antibodies)electrophoresis results showed that rHis-SseB and rGST-SseB portein were successfully expressed.The proteins were purified using His-tag purification column and GST-tag purification column after inducing expression of a large amount of protein.The SDS-PAGE electrophoresis results indicated that rHis-SseB and rGST-SseB with higher purity could be obtained by purification with 97%and 84%.Analysis of Western blot with multi-antibody against Salmonella enteritidis,respectively,showed that rHis-SseB and rGST-SseB had good immunoreactivity Stimulation with rHis-SseB of mouse macrophages can significantly induce the secretion of cytokines such as IL-1?,IL-4,IL-6,IL-12,IFN-?,TNF-?.The results indicated that the recombinant proteins rHis-SseB and rGST-SseB with good biological activity came out successfully through inducing expression,which provides important biological information for the development of Salmonella candidate subunit vaccines.2 Immune characteristics analysis and immune effect evaluation of Salmonella SseB protein candidate subunit vaccineIn order to evaluate the ability of Mn to enhance cell response induced by rHis-SseB,mouse peritoneal macrophages as a model.The results showed that compared with SseB protein stimulation group,its ability to induce cell secretory IL-4,IL-12,IFN-y after mixed with Manganese was significantly enhanced.Based on mouse experiments,rHis-SseB protein and Manganese adjuvant(SseB+Mn)were mixed to immunize 6-week-old BALB/c mice by intramuscular injection,and PBS group,SseB protein group,SseB protein plus Aluminum adjuvant(SseB+Al)group served as control.The immunization dose of rHis-SseB protein for each mouse was 30 ?g.The booster immunization was performed on the 14th day.The mice were bled orbitally at 12 days,21 days,and 26 days after immunization,rGST-SseB was used as the coating antigen,and the expression level of SseB specific antibody in serum was detected by indirect ELISA.The results showed that the level of SseB specific antibodies in the SseB+Mn group was markedly higher than that of the SseB group alone on the 12nd day(P<0.05),21st days(P<0.001),and 26th days(P<0.01).The detection results of IgG subclasses indicated that the SseB+Mn group can induce the production of antibodies of different IgG subclasses(IgG1,IgG2a).On the 21st day after the initial immunization,the splenic lymphocytes of mice were collected for cell immunity measurement.The results showed that the proliferation capacity of lymphocytes in the SseB+Mn immunized group was higher than that in the protein alone group.On the 28th day after immunization,the immunized mice were challenged with C50041 strain.The results showed that the immune protection efficacy of the SseB+Mn group was 40%,and the mice form SseB+Al group and SseB group were all died.The above results indicated that the use of rHis-SseB protein combined with Manganese adjuvant can induce mice to produce humoral immunity and cellular immunity,and enable mice to obtain immunoprotective efficacy to some extent.3 Immune characteristics analysis and immune effect evaluation of Salmonella multicomponent candidate subunit vaccineThe flagellin protein of Salmonella enteritidis C50041 strain was extracted by acid cleavage method.The results of SDS-PAGE showed that flagellin was extracted successfully.Western blot analysis on the extracted flagellin with anti-FliC monoclonal antibody as the primary antibody indicated that the extracted flagellin had good immunogenicity.To evaluate the ability of Mn to enhance rHis-SseB and FliC inducing cell response,use mouse peritoneal macrophages as a model.The results showed that compared with SseB and FliC stimulation group,its ability to induce cell secretory IL-4,IL-12,IFN-y after mixed with Manganese was significantly enhanced.Using mouse as animal model,rHis-SseB protein plus flagellin combined with Manganese adjuvant(SseB+FliC+Mn)was immunitied into 6-week-old female BALB/c mice.The PBS group,SseB+FliC group and SseB+FliC+Al group served as control.The immunization dose of protein for each mouse was 15?g rHis-SseB protein and 15?g flagellin at each immunization.Booster immunization was performed on the 14th day.On the 12nd,22nd days after immunization,mice were bled orbitally,and rGST-SseB and flagellin were used as coating antigens to detect the expression levels of SseB and flagellin-specific IgG antibodies in serum.The results showed that the level of SseB specific IgG antibodies in the SseB+FliC+Mn group was higher than that in the SseB+FliC group with statistical significance,at 12nd days(P<0.001)and 22nd days(P<0.05);flagellin specific IgG antibody levels in the group were higher than those in the SseB+FliC group in statistics at 12nd days(P<0.05)and 22nd days(P<0.01).On the 28th day after immunization,Salmonella enteritidis C50041 strain was used to challenge the immunized mice.The results showed that the immune protection effect of SseB+FliC+Mn group was 70%,and the SseB+FliC+Al and the SseB+FliC group were 40%and 30%,respectively.The above results indicated that rHis-SseB protein and flagellin combined with Manganese adjuvant can induce mice to obtain higher immunoprotective efficacy,which was significantly higher than SseB single component candidate subunit vaccine group.