Epitope Prediction And Its Application In Diagnostic Antigen Screening

Schistosomiasis is widely distributed and it does great harm to human health and livestock. Schistosome eggs or adult worms antigen have been used as antigens in the commonly immunological diagnostic methods. The components of these antigens are complex, and the supply is restricted. The recombinant antigens can get high purity but mostly in low sensitivity, which limit their wide application. Therefore, it is eager to get highly sensesivet, accessible antigens to replace the crude antigens or full-length protein in order to make the diagnosis more specific and sensitive.In this study, two proteins, which are SjRibosomal_L18ae (AY813148.AAW24880) and SjHABP4_PAI-RBPI (AY815608, AAW27340) have been selected by using the B cell epitope prediction software and bioinformatics analysis. The study for these two potential candidates include:(1) bioinformatics analysis,(2)the cloning of target genes and the expression and purification of recombinant proteins,(3)the immunogenicity analysis of recombinant proteins,(4)the stage-specific transcription profile,(5)the evaluation of diagnosis value of recombinant proteins and the synthetic epitope diagnostic value.SjRibosomal_L18ae is a ribosomal protein with binding multiple RNAs core domain in order to play capabilities, which belongs to the L18family. We constructed the recombinant plasmid of SjRibosomal_L18ae/pET-28a and got the recombinant SjRibosomal_L18ae with high purity. rSjRibosomal_L18ae is insoluble. SjRibosomal_L18ae shows higher transcription level in the every developmental stages tested.It is a housekeeping gene and plays an important role in the worms. The r SjRibosomal_L18ae can be recognized by both the patient and nomal serum in western-blotting assay. In ELISA assay using the rSjRibosomal_L18ae as antigens, it is showed53.3%and100%of sensitivity and specificity, respectively (15Positive serum samples and10negative serum samples). When the synthetic epitope P1and P2predicted from SjRibosomal_L18ae were used in the ELISA analysis, P1shows60%and100%of sensitivity and specificity, respectively (10Positive serum samples and10negative serum samples), while P2shows70%and100%of sensitivity and specificity, respectively.HABP4_PAI-RBP1family includes the HABP4family of hyaluronan-binding proteins, the PAI-1mRNA-binding protein and PAI-RBPl. HABP4has been observed to bind hyaluronan. PAI-1mRNA-binding protein specifically binds the mRNA of type-1plasminogen activator inhibitor (PAI-1), and is thought to be involved in regulation of mRNA stability. We constructed the recombinant plasmid of SjHABP4_PAI-RBP1/pET-28a and got a recombinant nSjHABP4_PAI-RBP1with high purity. It is insoluble. The transcription specificity analysis showed that schistosomula has the highest transcription level, sporocysts, male worms and eggs have a high transcription level, miracidia has a low transcription level. Female worms and cercariae didn’t detected the transcript. Both the patient and normal serum can recognized the nSjHABP4_PAI-RBP1. When the nSjHABP4_PAI-RBP1was usedas the antigens in the ELISA analysis, the sensitivity and specificity are60%and100%, respectively.