Immune Efficacy Study Of Important Membrane-Associated Proteins In Streptococcus Suis Serotype2

Streptococcus suis2(S. suis2) has had an impact upon the healthy development of livestock and the safety of human life health and property more and more severely, as an important zoonosis pathogen. Because of numerous serotypes, lack of cross-protection among different serotypes and bacterial strain variability, up to date, effective vaccines has not been found. Although commercialization of inactivated vaccines have been found, there are still many shortcomings, such as immune to the adverse event, a narrow spectrum of immune protection etc., it is badly in need of identification of candidate antigens with high immunogenicity and universal type, and studying immunity mechanism of them for further revealing the pathogenesis and immune mechanism of S.suis type2. In the research, six immunogenic membrane-associated proteins (SSU043、SSU0482、SSU0927、SSU1634、 SSU1637and SSU1853respectively) were choosed to study the immune efficacy of them. In order to elect vaccine candidate antigens with high immunogenicity and universal type, the recombinant proteins were studied about the immune efficacy through gene cloning, expression and purification of proteins, and laid the foundation of development of novel and effective vaccines. The primary researches content as follows:1. The cloning, expressing and purification of SSU0434、 SSU0482、 SSU0927、 SSU1634、 SSU1637and SSU1853Primer sequences were desigened referring to genome sequence, and gene segments were amplified from SC19strain (the homologies of target genes are100%), then prokaryotic expression vectors for SSU0434、SSU0482、 SSU0927、 SSU1634、 SSU1637and SSU1853were constructed. The vectors were transformed into E.coli DE-3after sequedcing and were expressed. If the proteins were what we need, the proteins were induced and expressed largely for the researches.2. Valuation of mmune protection force for six membrane-associated proteins SSU0434.SSU0482、 SSU0927、 SSU1634、 SSU1637and SSU1853BABL/C mice were immuned with the purified proteins, six mice in every group, and on the fourteenth day mice were challenged with SC19strain by intraperitoneal injection after the last immunization. The dose was2×109CFU for every mouse, and observed for fourteen days. The survival rate were83.3%,83.3%,33.3%,66.7%,50%and83.3%respectively. Because of higher protecting force, SSU0434, SSU0482and SSU1853were choosed out for the next immunogenicity and immune protection researches. 3.Valuation of immunogenicity and immune protection research for SSU043^SSU0482and SSU1853separately or assemblyThe results of Western-blot indicated that SSU0434, SSU0482and SSU1853could cause specific immune response in mice.Four-weeks old femal BABL/C mice were immuned with the purified proteins of SSU0434, SSU0482, SSU1853and SSU0434-SSU0482-SSU1853, and challenged after the last immunization, finally the survival rate were60%、50%,60%and40%respectively. Lymphocyte proliferation were conducted with MTS, measured at OD490nm. Proteins for SSU0434and SSU0482could make the lymphocyte from spleen of immuned mice proliferate significantly, while the protein for SSU1853and SSU0434-SSU0482-SSU1853could not make the lymphocyte from spleen of immuned mice proliferate significantly. The above lymphocyte were cultured for48-72h, and then the supernatant was collected to detect the levels of IFN-yand IL-6. Compared with the controls, the levels of IFN-yand IL-6increased significantly, the results indicated that Thl and Th2type immunoreaction occoured at the same time in immuned mice.SSU0434、SSU0482、SSU1853and SSU0434-SSU0482-SSU1853could stimulate mice to produce specific high degree of antibodies, and the level of IgG1and IgG2a had no significant difference, the results indicated that Thl and Th2type immunoreaction occoured at the same time in immuned mice.4. Passive protection assays for SSU0434、SSU0482and SSU1853separately or assemblyEvery mouse was injected100μL antiserum(SSU0434、SSU0482、SSU1853and SSU0434-SSU0482-SSU1853separately), eight mice for every group, challenged with SC19strain after24h and observed for fourteen days. The survival rate for SSU0434、SSU0482、 SSU1853and SSU0434-SSU0482-SSU1853were87.5%、75%、62.5%、50%respectively, the results indicated that the specific antibodies for SSU0434、SSU0482、SSU1853and SSU0434-SSU0482-SSU1853may make an impact in specific resistance anainst SS2.5. Bactericidal assays of antiserum for SSU0434. SSU0482and SSU1853separately or assemblyAntiserum for SSU0434、SSU0482、SSU1853and SSU0434-SSU0482-SSU1853(95μL), sterile PBS(95μL), blank mouse blood and SC19strain(105CFU) were coincubated for1h, and calculated the killing rate. The results indicated that antiserum may not play a role of killing, the reason was antibodies in antiserum ccould not react with bacrerium, while nonspecific components, such as phagocyte and neutrophile etc. in the blood play a role of killing bacterium.