Mining Potenyial Diagnosis Antigen Genes From The Transcriptome Data Of Clonorchis Sinensis And Their Expression And Evaluation

Objective To mine potential diagnostic antigens of Clonorchis sinensis(C.s.), the expression sequence tag (EST) database was screened for the genes of interest by bioinformatics analysis and the selected genes were expressed, the recombinant proteins purified and primarily evaluated for sero-diagnosis. Method 1. Screening candidate antigen genes with the method of bioinformatics. The EST database was screened using the Tandem Repeats Finder software for tandem repeat sequences, and the SignalP 4.1 software and others for secretory proteins; 2. Expression and purification of recombinant proteins. The screened genes were cloned into pET28a vector, induced by IPTG, and purified by His-bind-resin [(Ni-NTA) QIAGEN] affinity chromatography; 3. Primarily evaluation of the recombinant proteins. The recombinant proteins were examined and evaluated with the serum samples of infected persons for highly immunogenic antigens. Result 1. Twenty-two predicted secretary proteins were found,7 of them were known proteins, and 15 (classified into 6 categories) new; 2. The proteins families of Cs22X? Cs79? CsKY were successfully expressed in prokaryotic expression system, the Cs22X family proteins were soluble, while the Cs79 and CsKY insoluble; 3. The Cs22?Cs1299?Cs5B1 and Cs1687 proteins of the Cs22X family could distinguish clonorchiosis and healthy persons' sera. The sensitivities and specificities of PrCs22-2r and PrCs22-3r by ELISA both were 45.71% and 96.77%, showing no cross reactivity with schistosomiasis japonica and cysticercosis cellulosae sera, but only one case with 15 paragonimiasis westermani.4. Preliminary analysis showed that the antigenic determinants may be located in the tandem repeat sequences, and the Cs22 proteins were inferred into GPI-anchored proteins. Conclusion The method by screening tandem repeat sequences combined with the secretory protein prediction tried in this study was demonstrated to be an applicable approach in mining potential diagnostic antigens for clonorcihasis.