Preparation And Protective Efficiency Evaluation Of Outer Membrane Proteins And Outer Membrane Vesicles As Subunit Vaccines Against Salmonella Enteritidis

Salmonella is an important food-borne pathogen closely related to human salmonellosis and public health.Consumption of Salmonella-contaminated foods can cause human infection.Recently,Salmonella enterica serovar Enteritidis(S.Enteritidis)has increased to be top one serovar among Salmonella serotypes in causing human Salmonellosis.Since S.Enteritidis can be transmitted horizontally and vertically,it can spread along the food production chain "from farm to table".Therefore,control of Salmonella in the poultry industry is the fundamental measure used to prevent its spread in food production chain.Vaccination is the key method used to control avian Salmonellosis in poultry farm,which is also an effective measure to control its transmission to humans.Until now,there are some commercialized inactivated and attenuated vaccines used against non-typhoidal Salmonella used in animals,but few reported subunit vaccines.The DNA recombination technology has been used to obtain the pure antigen proteins or peptides as subunit vaccines with simple structure,safety,nontoxic side effect,and good protective efficiency,so the subunit vaccines are considered as one of the ideal vaccines.To develop subunit vaccines against avian Salmonellosis,the outmembrane proteins OmpC and OmpF with high immunogenicity were used as the targets.The OmpC and OmpF was expressed as inclusive body using prokaryotic expression system,and purified to be used as immunogen.Mixed with Fruend's adjuvants,the protective efficiency of two proteins was evaluated in HY-Line chickens,and different adjuvants were then used to evaluate the potential subunit vaccines based on analysis of protective efficiency,clearance of bacteria,titers of IgG,cytokines expression,etc.In addition,the OMV was extracted from S.Enteritidis C50041 to be used as another subunit vaccine candidate compared with OmpC and OmpF.All of these experiments were defined to provide materials and basis for development of outmembrane proteins or OMV as subunit vaccines against avian S.Enteiritidis1.Expression and purification of outermembrane proteins OmpC and OmpF from S.Enteritidis and extraction of Salmonella OMVThe ompC and ompF gene was amplified from S.Enteritidis reference strain C50041 using PCR and cloned into prokaryotic expression vector pET30a(+)to generate the recombinant expression plasmids pET30a(+)-ompC and pET3 0a(+)-ompF.After identification by PCR analysis and sequencing,both of the recombinant plasmids were transformed into the host strain E.coli BL21(DE3)to produce the recombinant bacteria pET30a(+)-ompC-BL21(DE3)and pET3 0a(+)-ompF-BL21(DE3),respectively.The recombinant outmembrane proteins OmpC and OmpF was expressed in the form of inclusive body at the induction of 0.6mM IPTG.The proteins were prepared as the subunit vaccine candidates after denaturation,renaturation and purification.In addition,the OMV was extracted from S.Enteritidis C50041 using ultracentrifugation,and followed with analysis of morphological structure by the transmission electron microscope.As the OMV has been considered as a new adjuvant in vaccine development,the extracted OMV was prepared to evaluate its immunogenicity and protective efficiency against S.Enteritidis.2.Protective efficiency of OmpC,OmpF and OMV as subunit vaccine candidates against S.Enteritidis infection in chicken.The purified OmpC,OmpF was mixed with Freund's complete adjuvant and/or OMV,respectively.The mixed substances were then injected intramuscularly into 7-day-old HY-Line chickens,followed with the second immunization at 14-day-old with the same antigens.The group immunized with the Freund's complete adjuvant was changed into Freund's incomplete adjuvant.Seven days later,the wild strain CZ14-1 isolated from poultry was used to infect chickens with 2.1×109CFU intramuscularly.The result showed that immunization of OmpC and OmpF protected 70%and 75%of chickens,respectively.While 80%and 100%of chickens survived in the group immunized by OmpC mixed with OMV and that of OmpF mixed with OMV,respectively.The PBS immunized group has only 25%of survived chickens.In addition,immunization of proteins or OMV showed no effect on the weight of chickens.In conclusion,the OmpF was better than OmpC to be developed as subunit vaccine,and OMV is better than the Fruend's adjuvant.To evaluate the ability of vaccine candidates to clear S.Enteritidis in chickens,2.0×109CFU of CZ14-1 was used to attack the immunized chickens.The result showed that Quil-A,Al(OH)3,and Fruend's adjuvant could protect 95%,85%,and 75%of chickens from death,respectively.The absolutely OMV immunized group offered 90%protective efficiency,while 30%of unimmunized group survived in the attack.No evident difference was detected among the immunized groups.Furthermore,the bacterial colonization was detected in each immunized group challenged with 2.0×108CFU of CZ14-1at 1,3,7,14,21,28,and 35 days post infection.The results showed that the clearance of Salmonella in organs and tissues was quicker in the group immunized with OmpF combined by Quil-A,and the OMV immunized group than the other groups,which showed complete clearance of bacteria in liver at 14 and 7 days post infection,respectively.Both groups displayed clearance of Salmonella in spleen at 14 days post infection.Indirect ELISA was used to detect IgM and IgG antibodies in serum.The results showed that the immunized group could produce IgM and IgG antibodies against OmpF in the sera at 7 days post immunization,and the antibody levels gradually increased after the boosting immunization.qRT-PCR analysis was used to detect the expression level of cytokines in spleen cells from each immunized group at 7 and 14 days post immunization.The results showed that the expression levels of IL-4,IL-6,and IFN-? were significantly up-regulated compared with the negative group Therefore,the OmpF and OMV has the potential to be developed as subunit vaccines against avian S.Enteritidis.