Study On Recombinant Protein Antigens Of Schistosoma Japonicum RPS4 In Diagnosis Of Schistosomiasis

BackgroundSchistosomiasis japonicum remains to be a serious zoonosis and mostly prevalent in developing country. In our country it mainly locates in 5 provinces around lake and 2 provinces at mountain area. Researches of rapid immunological diagnosis on schistosomiasis are significant for monitoring the source of infection and epidemic control. It is generally believed that the ideal diagnostic methods on spot should be specific, sensitive, cheap, convenient, stable and rapid. Nowadays the diagnostic methods are Kato-Katz, IHA(Indirect hemagglutination test)and ELISA (Enzyme-linked immunosorbent assay)yet. Kato-Kotz as a pathogenic diagnostic method is time costing and labor consuming with a poor positive recall rate which is easy to show false-negative in lightly epidemic areas, so it is not suitable for screening in a large area. Immune agglutinin method has lower specificity ,it was easy to show false-positive. Therefore, establishing new, fast, accurate, low-costing and convenient techniques or methods comes to be the key concerns in schistosomiasis diagnosis.ObjectiveThe recombinant expression vector pQE30/SjRPS4 was preserved by our laboratory, expressed in E.coli M15. forthermore , the recombinant protein was purified by Ni-NTA resin Fast Start column, the immunocompetence of the expressed protein was analyzed. Indirect ELISA were used to detect antibodies of human serum with schistosomiasis japonica in endemic areas. This lays the foundation for the diagnosis and epidemiologic research of schistosomiasis japonica.Methods1. SjRPS4 was expressed largely, purified and identified its diagnostic value. E.coli M15 containing with recombinant plasmid was cultivated largely, induced by IPTG, The recombinant protein was purified by Ni-NTA resin Fast Start column after renaturation and its concentrations and immunoreactivity were determined by BCA method and Western blotting.2. Development of Indirect ELISAIndirect ELISA was developed by coating microwell plates with the purified protein SjRPS4.Then the normal swach of positive or negative was established after optimized the working conditions for the indirect ELISA.3.Application of SjRPS4 Indirect ELISA in Schistosomiasis endemic area.The serum samples of patient infected by Schistosoma japonicum with suspicious was collected from Renyi village, Shuangyi village, Datang village, Mutun village, Baitang town, Miluo city. The serum samples were detected by Indirect ELISA and Indirect hemagglutination test (IHA) as reference method to assess the value of the recombinant protein in serodiagnosis.Results1. The SDS-PAGE demonstrated that the SjRPS4 recombinant protein with relative molecular weight about 30.96×10~3 was expressed after the E.coli M15 containing the recombinant plasmid was induced by IPTG, and mainly existed in the pattern of inclusion body. Its purity reached up to 95% after purification by Ni-NTA resin Fast Start column. The Western blotting proved that it could specifically react with Schistosomiasis japonica positive sera.2. The indirect ELISA used the purified recombinant protein as coating antigen was developed and optimized. The normal swach of positive or negative was 0.12. The indirect ELISA showed that no cross reaction with Paragonimiasis positive sera.3. The fellowing was the result of indirect ELISA diagnosis value in the Schistosomiasis endemic areas. The sensitivities and specificities of the indirect ELISA were 90.91%(70/77) and 92.59% respectively for the Schistosomiasis japonica suspicious patients sera. The concordance rate of between the indirect ELISA test and the IHA test to 104 patients sera was 91.35%.Conclusion1. The Recombinant protein SjRPS4 was espressed and purified by Ni-NTA resin ,it showed excellent immunoreactivity, and could specifically react with Schistosoma japonicum positive sera, and could be applied to Schistosoma japonicum serodiagnosis.2. The indirect ELISA method for detection of antibodies against Schistosoma japonicum ribosomal protein S4 was establisted, it showed excellent application value in serodiagnosis.