Schistosoma Japonicum Complex Epitope In The Synergy Of Cpg Odn-induced Th1 Response Protective Immune Function In Mice

Schistosomiasis is a serious threat to the public health worldwide. Over 100 million people in china are threatened. Eradication of the disease by interdiction of prevalence and transmission is the main aim of researchers who are engaged in the study on Schistosomiasis.The comprehensive control measure based on the chemotherapy of praziquantel, combined with snail control in epidemic areas has made great achievements for the control of schistosomiasis. There is a high priority for the development of effective anti-shistosomiasis vaccines in order to prevent schistosome infection and re-infection. Successful vaccine development for schstosomiasis has been hindered by lack of consensus on the type of immune response that would provided maximum levels of protective immunity and incomplete knowledge of the key anti-parasite effector mechanisms. Many vaccine studies conducted in mice indicate the importance of type-1-cytokine-mediated effector mechanisms that are highly dependent on the production of interferon - v (IFN- y ) from CD4 T cells, while acquired resistance in humans correlates with type-2-cytokine-mediated response that is mediated by IgE acting in conjunction with effector cells such as eosinophils and platelets. Recent finding indicates that a mixed Th1/ Th2 is highly effective as an immunization strategy. Therefore, we selected and identified T cell epitopes from some candidate vaccine molecules for preparation of multiple-epitope protein vaccine and multiple-epitope DNA vaccine. CpG ODN which can highly stimulate proliferation of mouse splenocytes was also selected and identified. The specific and highly polarized type-1-dependent response can be developed in vaccinated mice with the vaccine and specific co-stimulatory molecules CpG ODN or IL-12. The protective immunity of mice against the schistosome infection was examined extensively, and the significance of Thl response polarization to the development of anti-schistosome immunity was investigated.1. Cloning of Sj28GST gene and purification of its highly expressedproductIn order to clone Schistosoma japonicum 28kDa glutathione S-transferase(Sj28GST) gene from adult worm cDNA library and obtain expression product of the gene for selection of T cell epitopes and further vaccine study, the specific primers P1 with Nco I and P2 with Xho I restriction enzymes were designed according to Sj28GST gene sequence from GenBank. SJ28GST gene was amplified from adult worm (Jiangxi isolate) cDNA library by PCR. The PCR product was identified by agarose gel electrophoresis and purified by cutting the agarose gel containing the amplified gene product, then inserted into vector pGEM-T(Promega) to produce recombinant plasmid. The SJ28GST gene was amplified again by PCR using the recombinant plasmid as template and sequenced using primers PI and P2 on ABI 377 sequencer. The results indicated that SJ28GST gene was 633bp in length, coding for 211 amino acids as the same as those in GenBank. The purified PCR product and plasmid pET32c(+) were digested with Nco I and Xho I, respectively and ligated to construct the recombinant plasmid(pET32c(+)/Sj28GST). The recombinant plasmid Sj28GST/pET32c(+)/Sj28GST was approved to construct successfully, when the band , which is about 630 bp similar to Sj28GST gene in length, appeared in agarose gel by digestion of the recombinant plasmid pET32c(+)/Sj28GST with enzymes Nco I and Xho I. The recombinant plasmid pET32c (+)/Sj28GST was then transformed into E.coli BL21(DE3) and expressed by IPTG induction. The result from SDS-PAGE suggested that the fusion protein SJ28GST-TRX was expressed with the molecular weight of 43kDa. Western-blotting analysis showed that the expressed fusion protein could react with sheep anti-Sj28GST antibodies.A purification procedure for the recombinant Sj28GST-TRX expressed in Escherichia coli was carried out as follows to obtain this ideal fusion protein. The Sj28GST7pET32c(+)/E.coli BL21(DE3) was induced by IPTG to get large amount of the fusion protein. The cultured and induced E.coli was...