| Schistosomiasis japonica remains one of Zoonoses and prevalent in the south of China. To control schistosomiasis, it is necessary to adopt effective and safety vaccines against schistosomiasis. Therefore, to rationally and efficiently screen or identify protective molecules against schistosoma has been one of the hot and difficult tasks. Previous researches showed that high protective effects against schistosomiasis could be induced in various hosts immunizated with radiation-attenuated (RA) cercariae or schistosomula, the mechanism of this high protective effect was mediated by CD4 + Th1 cells in mouse model. In addition, since the surface of the parasite (tegument) contact directly with the host immune system, so some molecules from the tegument or secreted from worms could be the promising protective candidate molecules. Obviously, for the efficient and rational development of vaccine against schistosome, it is one direction of researches worth exploring to evaluate the issue that whether the molecules from the tegument are able to simulate the protective effect mechanism of the RA vaccine. Preliminary studies in our lab by Chen et al found that one molecule so called prosaposin from schistosoma japonicum (SjPSAP) was predicted to contain a signal peptide and may locate on the tegument, and one peptide within SjPSAP may be a potential T cell epitope. So we speculated that SjPSAP may be a novel protective candidate antigen against schistosomiasis. However, the problem that whether SjPSAP is located in schistosome tegument and is able to simulate the protective immunity induced by the RA vaccine remains to be experimentally validated. Therefore, in this study we further evaluated the immunobiology function of SjPSAP moleculae, aiming at providing the basis for efficient and rational discovery of the protective antigens against schistosome.First, SjPSAP was cloned and expressed with the molecular biology techniques. Westen Bloting method was used to analyse the immunogenicity of SjPSAP. Immunofluorescence was applied to observe the location of SjPSAP in parasites at various development stages. The results showed that the recombinant protein (rSjPSAP) was successfully cloned and expressed, consistent with the predicted molecular weight of 106kDa, although the expression quantity was very low. Western bloting analysis showed that the rSjPSAP could be recognized by the positive serum from mice infected with S. japonicum, and serum from mouse immunized with rSjPSAP protein could also recognize the counterpart molecule from parasite of 7d, 14d, 23d, 32d and 42d; In the immunofluorescence detection, green fluorescence signal were observed on the tegument of schistosomes at 7d, 14d, 23d, 32d and 42d. These results suggest that recombinant protein rSjPSAP has antigenicity, and SjPSAP occurs in worms at different stages of S. japonicum and is likely to be located on the body surface of parasites. These demonstrated that it is worth investigating the immunological function of SjPSAP.Secondly, in order to assess whether SjPSAP molecules has the capacity to simulate high protective effect mediated by CD4 + Th1 cells in the host induced by the RA vaccine. T helper cell epitope peptides within the SjPSAP were in silico predicted using the online tools; Firstly, we selected out 11 possible peptides binding to MHCII molecule in hosts using SYFPEITHI, MHCPred, HLAPRED, ClusPro and other online server ; then we immunized mice with these synthetic peptides, and found that 5 of the predicted peptide(sp75-89,p402-416,p904-918,p93-107,p16-30)could significantly stimulate the proliferation of immune splenocytes in vitro (stimulation index SI is greater than 2, and their SI were significantly different compared to the control group, P <0.05) by using the modified MTT method; the ELISA assay results showed that stimulation of six predicted peptides (p75-89, p809-823, p490-504, p904-918, p93-107, p16-30), together with the positive control peptide, could made IFN-γsecreted by spleen cells increased significantly; cytokine secretion profiles tests of CD4 + T cell from immune mice showed that the ratio of CD4 + T cells secreting Th1 cytokines IFN-γis higher than the ratio of those secreting Th2 cytokines, after in vitro stimulation of splenocytes from the mice immunized with p75-89, p402-416, p904-918, p93-107, p16-30 peptide; flow cytometry detection results of direct binding of mouse spleen cells in vitro showed that 62.62% -90.38% of the cells were marked by biotin labeled peptide, and the rate of inhibition at 67.94% -90.78% when detecting with the competitive unlabeled peptide, while rates of inhibition at 57.61% -88.79% and 35.55% -77.05% respectively, when incubated with anti-I-Ad and anti-I-Ed antibody. These results indicate that SjPSAP molecule may contain four Th1 cell epitope peptides, which can bind well with murine antigen presenting cells.Finally, we conducted an anti-schistosome protection experiments in BALB / c mouse model with rSjPSAP and 6 selected peptides (including the 4 potential Th1 cell epitope peptides identified above and 2 other peptide p809-823, p856-870). The results showed that mice immunized with rSjPSAP had worm reduction rate of 40.57% and 41.54% (P <0.05)and the egg reduction rate of 40.39% and 43.30% in liver; mice immunized with 4 Th1 epitopes had 26.37% ~ 28.75% and 26.25% ~ 28.69% of the reduction rate, respectively, 22.85% ~ 29.61% and 25.96% ~ 30.90% of the liver egg reduction rate; mice immunized with other two peptides (p809-823, p856-870) induced worm reduction rate of 16.87% ~ 18.57 % and 20% 20.81%, and the liver egg reduction rate of 15.59% ~ 20.17% and 16.27% ~ 20.72%. In addition, the ratio of specific antibody subtypes IgG2a and IgG1 in serum from mice immunized with rSjPSAP was on the rise, and the ratio of IgG2a and IgG1 changed in different tendency in serum from mice immunized with 4 Th1 epitopes, while the ratio of IgG2a and IgG1 with subtle changes in mice immunized with the other two peptides. All the data suggests that rSjPSAP and the four Th1 cell epitope peptide have some protective effect against Schistosoma japonicum, and the effect of rSjPSAP may be related to Th1 cell-mediated immunity.In conclusion, we cloned and expressed the SjPSAP molecule, recombinant protein rSjPSAP has antigenicity, and SjPSAP occurs in worms at different stages of S. japonicum and is likely to be located on the body surface of parasites; and characterized that SjPSAP molecule may contain four Th1 cell epitope peptides, which can bind well with murine antigen presenting cells; and found that rSjPSAP and the four Th1 cell epitope peptides have some protective effect against Schistosoma japonicum, which may be related to Th1 cell-mediated immunity. All these provide theoretical and experimental basis for discovering more protective T cell antigen or epitope in a reasonable and efficient way. |
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