| Objective: To look for a new vaccine candidate of S. japonicum (Chinese mainland strain) by the way of signal transduction intervention, the signaling protein Sj14-3-3 coding gene from cDNA of S.japonicum adult worms was amplified and cloned into prokaryotic expression vector to express the recombinant protein (rSj14-3-3). To research the localization of the Sj14-3-3 in the parasite with its Monoclonal antibody (McAb) derived from mouse. To prepare the rSj14-3-3 protein and DNA molecular vaccines to estimate the immuno-protection in mice challenged with cercaria, and to observe the cooperation of the recombinant peptides rSj14-3-3 and rSjGST proteins and their DNA as candidate vaccine and the effect of y 8 -T cells activated and amplified by Mtb against S. japonicum. Based on the studies above, to discuss the value of rSj14-3-3 protein in the diagnosis of schistosomiasis. Methods: The mRNA of S. japonicum was extracted to prepare for cDNA by RT-PCR, from which the Sj14-3-3 coding gene was amplified. Following ligation of the target Sj14-3-3 DNA fragments to expression vector pET28a, the fusion protein Sj14-3-3 with 6-His tag was expressed in E.coli BL21. The frozen sections of male and femal adult worms and 15-day old schistosomula were prepared to demonstrate the distribution of the 14-3-3 protein in the parasite with indirect immunofiuorescence (IIP). The protein and DNA of the 14-3-3 were prepared and purified through SDS-PAGE, electroelution, dialysis and alkali burstmethod, then BALB/c mice were immunized followed by challenging infection and the immuno-protection was evaluated. We also discussed the diagnostic value of Sj14-3-3 protein by indirect ELISA in schistosomiasis. Results: The size of PCR-created product was approximately 765bp. And the insert of pET28a-Sj14-3-3 digested with BamH I and Xho I was the same in length as the PCR product. With the positive clone induced by IPTG, the fusion protein was expressed in E.coli BL,21. The molecular weight of the protein is about 32.5kDa. the expression band can be recognized by the sera of rabbit infected with cercaria, the polyclonal antibody against 14-3-3 E , the sera of schistosomiasis patients and the sera against UV-irradiated cercariae. IIP showed that the 14-3-3 is widely located in the tegument, the subtegument, the muscle and the parenchyma of adult worm and 15d's schistosomulum. The results of the immuno-protective experiments showed that the worm reduction was 32.20 % in Sjl4-3-3+Freund's adjuvant group, 31.10% in SJ14-3-3+SJGST+ Freund's adjuvant group, 27.96% in Sjl4-3-3+Mtb group, 26.00% in Sj14-3-3+SjGST+Mtb group, 27.10 % in SjGST+Mtb group, 34.20% in Sj14-3-3 DNA grpoup, 32.40 % SJ14-3-3+SJGST DNA group, respectively; the number of eggs in liver tissue was reduced by 50.40%, 53.30 % ,51.10 %, 58.60%, 51.30%, 50.74%, 55.23%, respectively. Indirect ELISA showed that the rSj14-3-3 has high sensitivity and specificity in the diagnosis of acute or chronic schistosomiasis. It is of practical value. Conclusions: the 14-3-3 gene was successfully amplified, cloned and expressed. The localization of the natural 14-3-3 protein was demonstrated in different distribution of parasite. The molecular vaccine of Sj 14-3-3 could partially induce resistance to the infection with 5". japonicum in BALB/c mice, but no cooperation of rSj 14-3-3 with rSjGST was observed in animal model investigation. Y 6 -T cell activated and amplified by Mtb (a low molecular heat-stable polypeptide of Mycobacterium) play a role in infection of S. japonicum, and the effects were similar to the immune reactions induced by Freund's adjuvant. Indirect ELISA suggested that the rSj 14-3-3 protein, as arecombinant antigen, has a practical value in the diagnosis ofschistosomiasis.
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