| Schistosomiasis caused by schistosome, is a wide spread parasitic zoonosis that causes serious healthy problem to both human and animals.For half a century, in preventing the infection of schistosomiasis, our country have acquired great achievement,however, the sole wide efficient drug for schistosomasis can't avoid high rates of reinfection after mass treatment. Therefore, it is necessary to develop an efficient safe vaccine and new anthelmintic drugs against schistosome.Pairing between male and female worm of schistosomes is the key process for successful development of female maturation.Based on above characters of schistosomes,doing the research above molecular mechanism of schistosoma gender differential and interaction between male and female can help to reveal the mechanism of development of pairing and producing eggs.Two ESTs were searched in the schistosemula cDNA enriched library. Blast analysis SjNANOS had 67% similarity with Schistosoma mansoni(Sm) and SjMSP had 79% similarity with Sm. Based on bioinformatics analysis. The full length cDNA of SjNANOS were 721bp, and the Open Reading Frame for SjNANOS were 525bp, encoding 175 amino acid with a molecular weightof 21kD. Real-time PCR analysis from the worms of various stages of S. japonicum revealed that the mRNA level of SjNANOS was highest in the 18 days schistosomula, and in female is higher than that in male, The SjNANOS cDNAfragment was subclonedinto a modified expression vector pET28a(+) and transformed into E.coli BL21(DE3) cells, and the production of recombinant SjNANOS protein was analysed. In the presence of IPTG, the 21kDfusion protein was expressed in included bodies.Western-blotting revealed that the fusion protein could be recognized by the rabbit serum specific to Schistosoma japonicum adultworm antigen preparation.SjMSP had a full length cDNA with 1716bp, complete ORF containing 1065 nucleotides, encoding 355 amino acid with a molecular weight of 42kD. Real-time PCR revealed that the mRNA level of SjMSP was highest in the 18 days schistosomula, lower in 13 days worms and 23 days schistosomula ,Converse to SjNANOS,SjMSP is higher in male than that in female.Two recombinant proteins were purified and renaturaed. Animal experiment indicated that the vaccination of recombinant protein SjNANOS plus 206 adjuvant in mice led to 31.4% worm and 53.8% liver egg burden reduction respectively, when compared with those of the blank control. And the recombinant protein SjMSP induced 24.8% worm and 20% liver egg burden reduction. To analyse the function of SjNANOS gene, three siRNAs were designed according to the sequence of SjNANOS, (S144,S323,S396),the SjNANOS of RNAi parasites were inhibited by Real-time PCR and Western blot.The highest induced 88.0% reduction of mRNA expression in Schistosoma japonicum interferenced for 6 days. We use two different dsRNA deliveries and the result indicated that electroporation was better than soaking. The result showed that the RNAi effect also depend on the dosage of dsRNA. When add the highest dosage (40μg ) to the medium and cultured for three days, the transcript of SjNANOS could be reduced by 84%. The mice which were challenged about 100 cercaries were delivered dsRNA (S323) by dynamic injection into tain veins, and the result indicated the level of mRNA expression was reduced significantly. In the whole exprement,the growth and development and paring of parasites were inhibited too.We obtained the two novel genes SjNANOS and SjMSP for the first time, and animal experiment indicated that the two proteins could induced partial immunoprotection.RNAi indicated not only the transcript of SjNANOS could be reduced ,but had an influence on the pairing between male and female.All the results will assist us to understand more functions about SjNANOS and SjMSP, and provide the basis for screening new Schistosomiasis vaccine candidate molecules and drug targets.
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