Expression Protective Antigens Of Streptococcus Suis And Protective Analysis In Mice

Streptococcus suis(SS)is an important zoonotic pathogen distributing all over the word.Due to the widespread use of antibiotics,Streptococcus suis has developed drug resistance,and more effective prevention and control methods need to be found.The dominant serotypes of Streptococcus suis are SS2,SS7 and SS9 and commercial vaccines show a poor cross protection among the different serotypes.Therefore,it is necessary to develop a universal vaccine with high cross protection.The subunit vaccine is considered a choice for the universal vaccine.In this study,the sugar ABC transporter substrate-binding protein(S-ABC)and the phosphate ABC transporter ATP binding protein(P-ABC)that were considered the potential conserved antigens were selected based on our previous data and the B cell epitopes EP-1 and EP-2 were screened from S-ABC and P-ABC by bioinformatic analysis for evaluation the protective efficacy against Streptococcus suis infection.1.Expression of recombinant antigens.The cording sequences of S-ABC and P-ABC were obtained by gene cloning,and the gene sequences of EP-1 and EP-2 were synthesized chemically.The resulting sequences were inserted into expressing vector to generate recombinant plasmids pET28a-S-ABC,pET28a-P-ABC,pET28a-EP-1,and pET28a-EP-2.After validation by DNA sequencing,the recombinant plasmids were transformed into the E.coli strain BL21(DE3)for prokaryotic expression.The expressed proteins were purified and subjected to western blot analysis.The western blot data indicated that all recombinant proteins were recognized by swine serum positive for Streptococcus suis.2.Establishment of mice model for Streptococcus suis infection.BALB/c mice were inoculated intraperitoneally with SS2,SS7 and SS9,respectively and the time and number of dead mice were recorded accordingly for determining the medium lethal dose(LD50).The LD50 of SS2,SS7 and SS9 was 3.56×107 CFU,4.02×107 CFU and 7.88×107 CFU,respectively,as determined by the cumulative method.These data suggested that the mice model for Streptococcus suis infection were established.3.Analysis of protective efficacy against Streptococcus suis infection.BALB/c mice were immunized with recombinant proteins S-ABC,P-ABC,EP-1 and EP-2 twice and subjected to intraperitoneal challenge with 8LD50 of SS2,SS7 and SS9,respectively.Survival analysis indicated that the protection rates of S-ABC and P-ABC against SS2 challenge was 87.5%,while the protection rates of S-ABC and P-ABC were 50%and 62.5%for SS7 challenge,100%and 87.5%for SS9 challenge,respectively.However,the protection rates of EP-1 and EP-2 against SS2 were 25%and 12.5%,respectively.The protection rates for SS7 were 37.5%,respectively.The protection rates for SS9 were 25%and 12.5%,respectively.The protection rates were low.Analysis of the levels of antibodies,IL-4 and IFN-y showed that S-ABC,P-ABC,EP-1 and EP-2 were able to induce antibodies specific to the antigens and significantly high levels of IL-4 and IFN-y in sera,lungs and spleens in the immunized mice.In summary,S-ABC,P-ABC,EP-1 and EP-2 were expressed and the protection efficacy of these proteins were examined in this study.The resulting data indicated that S-ABC and P-ABC showed a relatively high cross protection against SS2,SS7 and SS9 infection,especially,a remarkable cross protection to SS2 and SS9 challenge.This study gained an insight into the development of subunit vaccine with cross protection against the different serotypes of Streptococcus suis infection.