| ObjectiveSchistosomiasis remains a major parasitic disease, with 200 million people infected and 779 million people at risk worldwide. Lack of effective vaccines and reliable diagnostic techniques makes this disease difficult to control. In an attempt to discover useful candidates for the diagnosis of schistosomiasis, proteomics in combination with western blotting were employed in this study. Furthermore, the host immune responses to these candidates were studied.MethodsFollowing two dimensional electrophoresis (2-DE), the adult worm fractions were transfered onto NC membrane for Western-blot analysis. The developed films were aligned and then matched with the homologous Coomassie brilliant blue (CBB)-stained gels by PDQuest software and artificial recognition. The matched spots were excised, digested by trypsin and further analyzed by LC/MS-MS. The potential biomarkers of the identified proteins were cloned and expressed from the transformed E. coli. The recombinants were purified, coated in micro-well plates and evaluated their performance in diagnosis of schistosomiasis using enzyme linked immunosorbent assay (ELISA). Besides, the BALB/c mice were challenged with the recombinant antigens and bled to obtain sera at different time points for specific IgG, IgG1 and IgG2a detection. The splenocyte from these mice were cultured in presence of the recombinants and analyzed for T-bet, INF-γ, IL-2, GATA-3, IL-4 and IL-10 using Real-time PCR, and the cultured supernatant were collected for INF-γ, IL-2, IL-4 and IL-10 cytokines detection.ResultsThis serological proteome assay yielded more than 30 immunodominant spots. Ten of these spots were precisely matched with a homologous two-dimensional electrophoresis (2-DE) gel and successfully identified by LC/MS-MS as corresponding to four different proteins. Of these proteins, SjLAP and SjFBPA were successfully expressed, and their recombinant protein products were further applied in the diagnosis of human schistosomiasis japonica using ELISA. The ELISA results revealed sensitivities of 98.1% and 87.8% for acute and chronic schistosomiasis with rSjLAP and 100% and 84.7% with rSjFBPA, whereas the assays showed a specificity of 96.7% with both recombinant proteins. After treatment with praziquantel, the titres of the antibodies against both antigens declined significantly (P<0.001) within 6 months post-treatment of praziquantel and were reduced to the control level between 6 to 12 months post-treatment in more than 80% patients sera.Anti-rSjLAP or anti-rSjFBPA IgG antibody was induced significantly higher at two weeks and reached the highest levels at six weeks after the immunization of BALB/c mice with either rSjLAP or rSjFBPA than the control group. And the anti-rSjLAP or anti-rSjFBPA IgG1 antibody was the same as its IgG antibody. However, the anti-rSjLAP or anti-rSjFBPA IgG2a antibody level was not significantly different after immunization. The cytokine profiles showed the higher levels of IL-4 and IL-10 were observed than the PBS control group in supernatants of spleen cells stimulated with rSjLAP or rSjFBPA. There was significant difference in the cytokine profiles of INF-γand IL-2 between the immunized mice and the control. Furthermore, the Real-time PCR determined rSjLAP or rSjFBPA significantly upregulated the mRNA expressions of GATA-3, IL-4 and IL-10 were but not T-bet, INF-γand IL-2.ConclusionSjLAP and SjFBPA were successfully screened and recombined from adult Schistosoma japonicum. Our present study confirmed that both rSjLAP and rSjFBPA were useful diagnostic antigens for S. japonicum infection and the mice challenged with both rSjLAP and rSjFBPA were provoked a robust Th2 immunodominance response. And the immunoproteomic approach has promising potential and offers a powerful tool in the screening of candidates for the diagnosis of schistosomiasis. Our findings may provide insight into schistosome diagnostic reagents and vaccines development. |
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