T Cell Epitopes Prediction Of Stage Specific Genes Which Screened From The Transcriptom Of Schistosoma Japonicum, And The Express Of The Schistosomulum Epitopes.

Schistosomiasis is one of the most important zoonosis, and it may be one of the most effective measures for controlling this disease to apply immunoprophylaxis to both animal and human being by using vaccine. Many studies of immune mechanisms in radiation-attenuated (RA) cercariae immuned mice models show that the high protective immunity is mediated by CD4+T cell. It would make a breakthrough for schistosome vaccine development if protective antigens or T cell epitopes could be screened out in RA mice models. Reverse vaccinology based on the principles of immuno-informatics and experiments, can not only screen out antigen molecules from the genome and transcriptom of pathogen in large-scale and high-throughput, but also save time and cost, and promise to be an effective tool to quickly identify the protective T cell epitopes of Schistosoma japonicum.Object:Aim at predicting the promiscous T helper cell epitopes of the candidated antigens screened out from stage-specific EST sequences of the Schistosoma japonicum transcriptom, constructing concatemers containing predicted multi-epitopes in the above candidated sequence from schistosomula stage, and expressing the concatemers of interest, eventually, providing basis for further experimental identification of the candidated Th cell epitopes.Method: Schistosoma japonicum transcriptom data was downloaded from database free available. Stage-specific EST sequences were screened out by calculating the numbers of each sequence in each stage of schistosoma japonicum, known antigen sequences were removed, then sequences were classified into two types of group, one group of sequences contains signal peptide and transmembrane structure, the other no-signal peptide and no-transmembrane structure. Sequences those were no-full length and not detected at the proteomic sequencing, were abandoned. Then epitope predicting severs were used to forecast epitopes of MHC classes I-Ad, I-Ed, I-Ak, I-Ek for antigen sequences. Peptides of each antigen were scored by web servers. The scoring of epitopes given in PRED-BALB/c server for MHC classes I-Ak, I-Ek was selected as basic condition, scoring got in SYFPEITH for MHC classes I-Ad, I-Ed and MHCPred, Rankpep for MHC classes I-Ad, I-Ed, I-Ak, I-Ek as reference, 15 epitopes with high score not only for I-Ak, I-Ek, but also for I-Ad, I-Ed in each server, were selected, five best promiscuous MHCП-binding epitopes were selected from each group of sequence in each stage. Concatemer containing five epitopes from both above two types of group, mep1 (group of signal peptide/transmembrane structure) and mep2 (group of non-signal peptide/transmembrane structure), sequences of the schistosomulum stage were designed by linking two glycins(GG.) between epitopes, respectively. The nucleotide sequences of above concatemers, with connected three protective bases(CGT), cleavage site BamHI(GAATTC), kozac sequence(CCGACC), start codon(ATG) and anti-malposition protective bases(GCT) at initiated end, with connected terminal codons(TAA), cleavage site HindⅢ(AAGCTT) and three protective bases(CGT) at the terminal end, were synthesized, respectively, and coloned into vector pUcm-T, then sequences of interest from vector pUcm-T were cloned into prokaryotic expression vector pET-28a(+), the expression of the concatemers of interest were induced by IPTG, products of expression were purified by His Bind Resin chromatographic column.Result: 166 stage-specific sequences were screened out from 8416 sequences of the schistosoma japonicum transcriptom, and 40 promiscuous MHCП-binding epitopes with high scoring were obtained from above sequences in all, The best five epitopes were obtained from each stage in each group. Both pET-28a(+)-mep1 and pET-28a(+)-mep2 were successfully constructed; The result of SDS-PAGE shown that pET-28a(+)-mep1 could be effectively expressed in BL21, pET-28a(+)-mep2 couldn't be expressed at all, A high concentration of expressed product of pET-28a(+)-mep1 was also purified. It could provide basis for further experimental identification of predicted T helper cell epitopes.