Research Of Schistosoma Japonicum Eggs And Associated Antigens

Schistosomiasis is a significant parasitic zoonotic disease. The diagnostic method inlaboratory to detect schistosoma infection played an important role in the prevention andtreatment of schistosomiasis. Currently, egg of Schistosoma japonicum was still the maindiagnostic antigen, while the extraction of eggs and the effective components of solubleegg antigen(SEA) were still inadequate.This study developed a new method for rapidly extraction of eggs firstly.We used thismethod to obtain the eggs, prepared the SEA and detected the feasibility as a diagnosticantigen. Then we used immunoaffinity methods to collect SEA which can specific bind toIgG and identified the components of it. At last, one antigen gene SjP40 was selected fromthe identified components to clone and expression. Then detected its value as a diagnosticantigen.Rabbits were infected with cercariaes and their livers were collected afterwards. Tofind the optimal condition in collected Sj. Eggs, the livers were homogenized and treatedwith different concentrations of NaOH at different times in different temperature. Thesensitivity of SEA as diagnostic antigen was determined by ELISA, which detection withpositive serum of schistosomiasis. Total 75 positive and 60 negative buffalo serum sampleswere tested to determine the reactivity of SEA extracts; With this kind of SEA developedthe indirect blood coagulation reagent which used for detection of schistosomiasis. Result,the optimal conditions to isolate eggs from livers were used with 5%~10% NaOH in 45℃for 5~10min; the yield of eggs was almost three times more than conventional meshmethod; the detection sensitivity of treated with NaOH which were higher than the SEAextracted from eggs by mesh method; the results of ELISA showed that the sensitivity andspecificity were 93% and 100%, which were 5% and 3% higher than the SEA obtained bymesh method; the indirect blood coagulation reagent has a good detection effect on rabbitand rat serum.In order to get the effective information of diagnostic antigen from SEA. From theserum of schistosome infected BALB/c mice, purified IgG was obtained and combined toresin. The components of SEA which can specific binding with IgG were collected byimmunoaffinity chromatography. This part of SEA was identified by mass spectrometry,and the result were analyzed using glycoprotein analysis and bioinformatics analysis. Theantigens which can specific binding with IgG remained good antigenicity; the results ofmass spectrometry show that there were 262 proteins included 76 protein groups;bioinformatics analysis revealed that these proteins were mainly involved in metabolicprocesses, biological regulation, response to stimulus and the like; these proteins had theactivity of binding and catalytic, electron carrier, enzyme regulation and other relatedfunctions; Dot-blot/SDS-PAGE with glycoprotein staining showed SEA fromchromatographic fractions contain various glycoprotein, the molecular weight distributionranging from 37-170 kd. We used an online prediction software chromatographic fractionspredicted the N- terminal glycosylation of SEA, there were totally 57 protein group havingN- terminal glycosylation sites, and these glycoproteins may be mainly involved singaling,immune system process, response to stimulus, locomotion and other process.According to the identification information of SEA, We selected SjP40 protein as theresearch object. The fragment of the full-length SjP40 gene was obtained by PCR, TheSjP40 was successfully cloned in pET-28a(+) and expressed in Escherichia coliBL21(DE3) cells and rSj P40 was expressed and existed mainly in the form of solublecondition. Western blot showed the rSjp40 had good antigenicity and immunogenicity. Theresult of testing different phase of the serum showed that rSjP40 had the value in earlydiagnosis.