Research Of Streptococcus Suis-Haemophilus Parasuis-Actinobacillus Pleuropneumoniae Triple Subunit Vaccine

Streptococcus suis(SS)is a common infectious disease in pig industry,which mainly causes septicemia,meningitis and arthritis in pigs.Haemophilus parasuis(HPS)and Actinobacillus pleuropneumoniae(APP)are the main respiratory infectious diseases of pigs.In this study,the protective antigen EF of SS and the protective antigen OMP,Apx I and Apx II of APP were cloned into pET-28a and pCold-sumo vectors by genetic engineering,and then transformed the vectors into E.coli BL21(DE3)respectively to construct recombinant expression strains.Afterinduced expression and purification,the recombinant proteins rEF,rOMP,rApx I and rApx II were obtained.Next,6 recombinant expression strains constructed in ourlaboratory(pET-28a-sly/BL21,pET-28a-mrp/BL21,pET-28a-oppa/BL21,pET-28a-oppa2/BL21,pET-28a-afua/BL21 and pET-22b-cdtb/BL21)were used to express and purify rSLY,rMRP of SS and rOPPA,rOPPA2,rAfu A,rCdt B of HPS.The above 10 proteins were equally mixed and emulsified with ISA 201VG to produce triple vaccine including 20μg perprotein in one dosage.AfterC57 mice were infected by 464 strain of serotype 2 SS(SS2)and GZ2 strain of serotype 9 SS(SS9)at different dilutions respectively,BALB/c mice were infected by HN10 strain of serotype 5 HPS(HPS5),ZD12 strain of serotype 13 HPS(HPS13),MD strain of serotype 5 APP(APP5),and S-8 strain of serotype 7 APP(APP7)at different dilutions respectively,the minimum lethal dose(MLD)for6 strains to mice was calculated.20 C57mice and 40 BALB/c mice were divided into two groups(10 C57 mice and 20 BALB/c mice in each group)to immunized with 300μL PBS+ISA 201VG and triple vaccine twice every 14 days respectively.The serum of mice was collected 14 days afterthe secondary immunization,and the antibody levels of EF,MRP,SLY,OPPA,OPPA2,Cdt B,Afu A,OMP,Apx I,and Apx II in each groupwere detected by indirect ELISA.28 days afterthe first immunization,C57 mice were divided into 2 groups from different immune groups,and challenged with MLD of SS2 and SS9 respectively;similarly,BALB/c mice were divided into 4 groups,and challenged with MLD of HPS5,HPS13,APP5 and APP7respectively.The results showed that:the MLD of SS2 and SS9 on C57 mice was 5×10~7 CFU,and that of HPS5,HPS13,APP5 and APP7 on BALB/c mice were 1.5×10~8 CFU,5×10~7 CFU,1.5×10~8 CFU and 7.5×10~8CFU respectively.Afterthe second immunization,the antibody levels against EF,SLY,MRP,OPPA,OPPA2,Cdt B,Afu A,OMP,Apx I,and Apx II in the experimental groupof mice were significantly increased(p<0.0001),while the control groupdidn’t changed.And the antibody levels between the experimental and control groups were significant different(p<0.0001).In the challenge experiment,mice in control groups all died within the observation period.The survival rate of mice in experimental groups to SS2,SS9,HPS5,HPS13,APP5,and APP7’s challenge were 100%,80%,60%,80%,80%,and 100%respectively,which were significant different from the control groups(p<0.05).All the above results indicated that the triple vaccine exhibited good cross protection against SS2,SS9,HPS5,HPS13,APP5 and APP7.This study is the first systematic research to verify the good protective effect of the triple vaccine,which provides an experimental basis forfurtherresearch and application.