Development Of Multi-epitope Subunit Vaccine For Streptococcus Suis

Streptococcus suis is an important zoonotic pathogen,among which Streptococcus suis serotype 2(SS2),serotype 7(SS7)and serotype 9(SS9)is particularly threatening to the development of animal husbandry and public health.Veterinary clinics usually use antibiotics to treat S.suis infection,but due to the unreasonable use of antibiotics,the resistance of Streptococcus suis is increased.Therefore,the use of vaccines remains the most effective strategy for the prevention and control of Streptococcus suis infection.However,the inactivated vaccine against Streptococcus suis has a large dosage and poor cross-protection.It is urgent to develop a safe and efficient universal Streptococcus suis vaccine.Therefore,this article has carried out epitope subunit vaccine of Streptococcus suis.In order to screen and identify the B cell dominant epitope protein of Streptococcus suis,we use bioinformatics software such as TMHMM,SignalP,ABCpred,BepiPred 2.0,Sopma and DNASTAR Protean to predict B cell epitopes of three important Streptococcus suis protective antigens glyceraldehyde-3-phosphate dehydrogenase(GAPDH),Muramidase released protein(MRP),Dihydrolipoamide dehydrogenase(DLDH),and finally 15 B cell antigen dominant epitopes were obtained.The recombinant plasmid GMD-pET-28 a was constructed by ligating the epitopes in tandem with the GGGG flexible fragment.The recombinant plasmid was transformed into E.coli BL21 to induce expression.After purification by nickel column and endotoxin removal treatment,a multi-epitope tandem protein GMD with a protein size of 43.3 kDa and a concentration of 3.34 mg/mL was finally obtained.In order to explore whether multi-epitope tandem protein GMD can protect the body against Streptococcus suis infection from different serotypes,GMD protein was immunized to mice and zebrafish respectively,and the immunoprotective effect of GMD on SS2,SS7 and SS9 infection was detected.After three GMD protein immunizations in mice,it was detected by indirect ELISA that GMD protein induced high levels of specific antibodies against SS2,SS7 and SS9 in mice.On this basis,the zebrafish was used for the immunoassay of GMD protein.After two immunizations,SS2,SS7 and SS9 were used respectively to attack.The results showed that GMD protein can provide good cross-protection ability for SS2,SS7 and SS9 infection.In order to enhance the immunoprotective effect of GMD protein,the mice immunoprotection test was carried out by using GMD protein combined with Toll-like receptor 4(TLR4)agonist MPLA(GMD+MPLA).Studies have shown that MPLA can act as a class of vaccine adjuvants that stimulate strong,non-specific immune responses in piglets.The experimental results show that GMD+MPLA-immunized mice can secrete a large number of specific antibodies,which can completely resist Streptococcus suis infection,and the immune protection rate reaches100%.The results of serum cytokine assay in mice after immunization showed that MPLA can simultaneously stimulate TH1-type and Th2-type T-cell immune responses,which can not only help B cells produce antibodies,but also activate key factors of cellular immune response.It is worth mentioning that the GMD+MPLA vaccine is less irritating and safer than mice in the whole-bacterial inactivated vaccine group.In summary,this study first used epitope analysis software to predict and acquire 15 B cell dominant epitopes of GAPDH,MRP and DLDH.Through zebrafish immunoprotection experiments,it was proved that GMD protein can protect the body against SS2,SS7 and SS9 infection in vivo.Further,through the mouse immunoprotection test,it was proved that the use of GMD combined with MPLA adjuvant can significantly improve the survival rate of mice,achieve 100% immunoprotective effect,and no adverse reactions.This study provides basic experimental data and new ideas for the development of a safe and efficient universal Streptococcus suis epitope subunit vaccine.