Immunogenicity Identification Of Predicted Outer Membrane Protein And Type â…£ Secretory Pathway Virb4Component Of Streptococcus Suis Type2

Streptococcus suis is an important zoonotic pathogen that causes a number of pathological conditions, such as fever, meningitis, endocarditis, arthritis, pneumonia, septicemia and so on, it severely threatens the practitioners working in the field of animal husbandry, and also causes serious diseases like meningitis and Streptococcal Toxic shock syndrome(STSS). Acording to the difference in the antigenicity of its capsular polysaccharide, it was divided into35serotypes and several other strains, which couldnot be stereotypied. Among them, the serotype2has always been considered the most virulent and the most frequently isolated serotype from diseased animals. In this study, S. suis2as the research subject, the possible virulence Omp40protein and VirB4protein were studied to provide a base for further research on the subunite vaccinate.1. Expression of predicted outer membrane protein and type Ⅵ secretory pathway VirB4component of Streptococcus suis type2The predicted outer membrane protein Omp40and Type VI secretory pathway VirB4component were putative virulence factors discovered through suppression subtractive hybrdization (SSH) between virulent strain HA9801and avirulent strain T15of SS2. According to published gene sequences of SS2human strain98HAH33’s Omp40and VirB4, two pairs of special primers were designed to amplify one part of gene respectively by polymerase chain rection(PCR) with the template of HA9801strain’s genome DNA and the products were subsequently inserted into the expression vector pET-32a(+).The recombinant plasmid was verified by nucleotide sequencing and transformed into E. coli BL21(DE3).Two fusion proteins were expressed in BL21after induced by IPTG, SDS-PAGE and Western blotting analysis showed that recombinant protein was about52.5kDa、56.4kDa, and positive reaction with the antiserum against ZY05719.2. Immunization Trials of predicted outer membrane protein and type VI secretory pathway VirB4component in ICR mice Blood was collected at particular times from ICR mouse, vaccinated with expressed Omp40and VirB4proteins independently or equivalently mixtured. The antibody titers of blood samples were analyzed by indirect ELISA and the expression levels of IL-4and IFN-y by fluorescent quantification PCR. The results showed that the antibody titer got the top14days after the second vaccination; all immune parameters of Omp40groups were significantly different from those of the blank control groups (P>0.05) and those of VirB4groups are almost similar (P<0.05); the Omp40and VirB4group increased in IL-4(P>0.05) but had no obvious change in IFN-γ(P<0.05).3. The immunogenicity and protective efficacy of predicted outer membrane protein and type Ⅵ secretory pathway VirB4component in zebrafish model350tailes of zebrafish were divided into5groups randomly:group1,2and3were vaccinated with purified Omp40, VirB4and balanced-mixture, respectively, and group4and5, as the control groups, with SS2HA9801inactivated vaccine and sterilized PBS, respectively. Booster with the same dose was taken14days after primerly vaccination and HA9801strain was injected with5O×LD5O/tail. The results showed that the protective rate of group1,2,3and4were22%,10%,38%and64%respectively while group5all died. Omp40and VirB4showed immune protective potency in zebrafish and that’s the base of subunit vaccines in the future.