Screening,Identification Of Schistosoma Japonicum Diagnostic Antigens And Its Preliminary Evaluation

Schistosomiasis is a serious, poverty-related public health problem in endemic countries. Sensitive diagnosis is the effective way to determine infection which plays an important role in the improvement of control and prevention strategies for schistosomiasis. Moreover, some strategies are built on the results of diagnosis, such as individual and community treatment, monitoring the epidemic situation changes, assessment of chemotherapeutic interventions and evaluation of epidemic trend. Traditional parasitological method is a reliable and classical approach for diagnosis of schistosomiasis. However, it is labor-intensive and time-consuming along with low sensitivity which would result in under-estimation in prevalence and infection intensity, particularly in the areas with low prevalence or after intervention. Therefore, the test is not an ideal field method for large-scale control of schistosomiasis.Immunological diagnosis would yield a higher sensitivity, specificity and practicality. This method is applied widely to control and prevention the disease and to evaluate the chemotherapeutic effect. The study of diagnostic antigens is the core field of immunological diagnoses because most methods are based on a good antigen.In this study, the adult worm and egg cDNA libraries were immunoscreened with the pre-treament and post-treatment serum samples from schistosomiasis japonica patients to obtain promising targets for detection or assessing therapeutic efficacy. For the first round, 120 positive clones were screened from the cDNA libraries. According to response intensity, categories and the length of inserts, we selected 17 positive clones to sequence the inserted fragments. Then, the DNA sequences were analyzed by bioinformatics tools.Based on the analysis results,10 target genes were selected for cloning and expression. The proteins which encoded by the genes were predicted their structures and functions. Positive clones SJA4-6, SJA6-5, SJE18-4, SJE20-4, SJE22-1, SJE22-4, SJE28-2 and SJE28-6 were used as templates to amplify the gene segments and construct 10 recombinant plasmids successfully. The recombinant plasmids were transformed into E.coli BL21. After the induction by IPTG,9 fusion proteins were espressed. There were 5 recombinant proteins having the soluble components and 4 recombinant proteins forming inclusion bodies. Through the affinity chromatography,4 recombinant proteins were purified from the bacterial lysate. Finally, we obtained rSjP1, rSjP2, rSjWSClP and rSjEFCAB products with high purity. The diagnostic values of purified recombinant proteins were evaluated by immunologic assay. An indirect enzyme-linked immunosorbent assay (ELISA) which used rSjPl, rSjWSC1P and rSjEFCAB as coating antigen respectively, could distinguish the pooled sera of normal persons from the pooled sera of schistosomiasis japonica patients. The optical density value of patient sera was 2.2-3.9 times higher than normal sera. When used rSjEFCAB as coating antigen, the ratio of OD value of the patient sera and the normal sera (P/N) was 3.9. The results showed that recombinant protein rSjEFCAB had promising application foreground.The sensitivity for detecting patients infected with Schistosoma japonicum by the indirect ELISA which used rSjWSC1P and rSjEFCAB were 72% (36/50) and 74% (37/50), respectively. The false-positive rates of detecting sera from normal people were 0-3.3% (0/30-1/30). When testing sera from patients infected with Paragonimuspulmonanis, Cysticercus cellulosae, Trichinella spiralis and Clonorchis sinensis by this method, the cross reaction rate was not observed in paragonimiasis,10%(1/10) in cysticercosis,11%(1/9) in trichinosis and 0-20%(0/5-1/5) in clonorchiasis. The results proved that recombinant proteins rSjWSC1P and rSjEFCAB had comparative higher sensitivity and specificity in detecting schistosomiasis. These recombinant proteins may be potential diagnostic antigens for schistosomiasis japonica.We also used immunoprecipitation to isolate the circulating antigen (CA) from schistosomiasis japonica patients' sera. Then the antigens were identified by LC-MS/MS and advanced bioinformatics tools to find high abundance antigen molecules. Because of the great genetic diversity between avian and mammalian animals, the antibodies from avian showed litte interaction with human's. In this study, the egg yolk immunoglobulin (IgY) against adult worm antigens were prepared and purified from immunized egg yolk. We developed a novel method based on IgY for identification and profiling CA in serum of schistosomiasis patients' sera. The IgYs against adult worm antigens were used as the capture antibodies to enrich the CA through immunoprecipitation, then the CA was determined by proteomics analysis. There were 7 proteins, including syntaxin 1 A, heterogeneous nuclear ribonucleoprotein H', carbonyl reductase 1, leucine carboxyl methyltransferase 1, fatty acid binding protein variant A, SJCHGC05715 protein and SJCHGC06828 protein, were identified as the components of CA. This method would be a new approach for further study of CA and lay a foundation for early diagnosis and evaluation of chemotherapy based on IgY and CA. This study presented the profile of schistosome CA which is essential for further development of diagnostic reagents for schistosomiasis.In conclusion, this study has provided scientific basis for the screening and identification of the specific diagnostic antigens for detection or assessing therapeutic efficacy of schistosomiasis japonica, and for future development of the relative technique.