| Schistosomiasis caused by Schistosome infection is a zoonotic parasitic diseases and a serious hazard to human health. Despite the availability of a highly efficient drug and the mass control programs in these years, schistosomiasis remains a significant public health problem in china because of the existence of intermediate host snails,livestocks,and re-infections.It can be predicted that long-term protection afforded by vaccination will be necessary for the full control or elimination of schistosomiasis. The result of proteomic analysis of schistosomula from hosts with different susceptibility shows that, schistosoma japonicum thioredoxin peroxidase Ⅲ(SjTPx-3) gene expressed highly in the BLAC/c mouse more than in Wistar rat or in Microtus fortis, and was found its distribution in the schistosome tegument, which suggested that SjTPx-3maybe play an important role in schistosome growth and development process and confrontation of the host immune attack.In this study, SjTPx-3gene was cloned by PCR based on GenBank GI. FN317765.1. sequence analysis shows its Open Reading Frame were663bp, encoding221amino acids with a molecular weight of24996.86Da, and isoelectric point of8.237. SjTPx-3had90%similarity with SmTPx-3in Phylogenetic analysis. Bioinformatics analysis showed that the less-hydrophilic protein had secondary structure of the supercoiled, and the possible transmembrane region at No.50-80amino acids with a strong antigenicity.,The SjTPx-3fragment was subcloned into the prokaryotic expression vector pET-28a(+) and pET-32a(+) using BamH I、Xho, I successfully to construct the recombinant expression plasmid pET-28a(+)-SjTPx-3and pET-32a(+)-SjTPx-3. Then two plasmids were trandformed in E. coli B121(DE3), the recombinant pET-28a(+)-SjTPx-3can be purified in two forms of soluble and inclusion bodies, and pET-32a(+)-SjTPx-3in form of inclusion bodies. Western blotting analysis with mouse serum immunized with SjTPx-3and rabbit serum Schistosoma japonicum adult worm antigen preparation showed that the two recombinant proteins have good immunogenicity and antigenicity.Real-time PCR analysis and Western blotting were employed to detect the levels of gene transcription and protein expression of parasites at different developmental stages. The results showed that, the mRNA level of SjTPx-3was highest in the35days adult worms and in female is higher than that in male, while it was transcriped in all detected stages. Immunohistochemical results revealed that, SjTPx-3located in the tegument of Schistosoma japonicum and the insect body. Aanti-oxidative activity of SjTPx-3was first demonstrated in the metal-catalyzed oxidation system (MCO).In this paper, the protective efficacy and the immune response induced by the recombinant protein, pET-28a(+)-SjTPx-3and pET-32a(+)-SjTPx-3, formulated with ISA206adjuvant were investigated in BALB/c mice. the results showed that A significant reduction in the worm burden and liver egg (37.02%, P<0.05and56.52%, P<0.05) was obtained in mice vaccinated with pET32a(+)-SjTPx-3, while pET28a(+)-SjTPx-3got13.04%and31.21%. the results indicated that the recombinant protein with the development of anti-schistosomiasis vaccine.ELISA results showed that specific IgG, IgG1,IgG2a antibody levels of mice vaccined elevated rapidly and remained, and the IgG1/IgG2a ratio also increased. The concentration of serum IL-2,IL-4、IL-10, IL-12and IFN-g(E) from mice immunized with rSjTPx-3plus206adjuvant were significantly higher than that in mice vaccinated with206adjuvant plus PBS. The results suggested that immunization with rSjTPx-3induced a mixed Th1/Th2response in which Th2was dominant.We systematically carried out the study of SjTPx-3, to explore its characterization and evaluate the potential as a vaccine candidate against schistosoma japonicum. All the results will assist us to understand the functions about the SjTPx-3, and provided the data for screening new schistosomiasis vaccine candidate molecule. |
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