The Prediction And Identification Of T Helper Cell Epitopes In Sex-differentially Expressed Molecule Of Schistosoma Japonicum

Schistosomiasis, caused by shcistosome, is a pervasive zoonsis with seriously threatened the human and animal health. Schistosome is gonochorism rarely seen in trematode fluke, and sexuality differentiation plays important role in its growth and pathogenesis. Pairing of the male and female worms is the key factor for female worm maturity and oviposition.Oviposition by mature female worm is a prerequisite for the spread of schistosomiasis as well as pathological damage in hosts.In the present study, based on the fact that the immunological effect mediated by T helper cell is probably the main factor in the vaccines of anti-fecundity and anti-pathogenesis against schistomiasis, aims to predict candidated T helper cell epitopes in the sex-differentially expressed genes screened out from the sequences of Schistosoma japonicum transcriptome; meanwhile, to validate previously-predicted T helper cell epitopes in schistosoma secretory molecules by lymphocyte proliferation assay. provide basis for further experimental identification of predicted T helper cell epitopes.Sex-differentially expressed molecules were screened out from the sequences of Schistosome japonicum transcriptome using digital differential display based on a statistical method of comparison-The Fisher Exact Test. The sequences screened out were analysed employing annotation data from both originals and the related sequence information of Chinese National Human Genome (South) Center; the signal peptide and transmembrane structure of the above sequenses were predicted using prediction software of protien secondary structure based on artificial neutral network ,HMM and weight matrix; The peptides of the above sequences binding to the MHCâ…¡molecules of human and mice were predicted using many severs based on binding motif, matrix , artificial natural network; The binding of above peptides to MHCâ…¡molecules was further optimized using docking simulation sever of receptor and ligand based on molecular dynamic algorithm. Five previously-predicted candidate T helper cell epitopes in Schistosome japonicum secretory molecules were synthesized artificially (coupled with the poly-lysine skeleton) .Proliferation of spleen lymphocyte in mice vaccinated with candidated epitopes was validated using MTT and ~3H-incoporated methods.The results showed that 20 full length sequences of sex-differentially expressed molecule of Schistosoma japonicum were screened out, of which nine genes expressed highly in male worms, the others expressed highly in female worms; seven of them were found to be secretory protein in the secondary structure prediction of the sequences (two were male highly-expressed, five were female highly-expressed), while the others were non-secretory ones; 21 T helper cell epitopes binding to MHCâ…¡molecules of human and mice were obtained by epitope prediction. Six promiscous candidated T helper cell epitopes were ascertained by optimizing the simulating predition of stucture docking, half of them from male highly-expressed ones. Including CSPYFVICTTKQYTR ( AY814326, secreted fictitious protein molecules of female worm) , SETLIQVWAVNKSVP (AY815950, molecules of ubiquitin thiolesterase activity of female worms) , EGSSVLVQRTHKSTS (AY810358, molecules of calpain activity of female worms) , YLCFVFGIIQAIYIF (AY813676, secreted sj25 molecule of tetraspanin superfamily of male worms) , FGLKLKRRDLIKDTM (AY812918, molecules of legumain activity of male worms) , and AEPIRMILVAAGVEF (AY816103, molecule of similar glutathione-s-transferase of male worms) .The validating result of five previously-predicted epitopes showed that two peptides epitopes, WQSIGYKFHRHGYE and STGFYLCTARSSLGS, could stimulate proliferation of lymphocyte, and the fomer one shown higher effect as judged by stimulating index.In conclusion, the present study have provided preliminary prediction in silico and the experimental validation basis for the further study on screening out the candidate Th cell epitopes in related molecules from Schistosoma japonicum, including screening out the potential T cell antigen molecules or epitopes of anti-fecundity and anti-pathogenesis.