Proteomics Of Adult Worm Soluble Antigen Of Schistosoma Japonicum With Mass Spectrum Identification And Bioinformatics Analysis

Objective:To Establish and optimize the methods of proteomics of adult worm soluble antigen of Schistosoma japonicum with mass spectrum identification, search for specific adult worm antigen inducing immunologic response from soluble protein componant. Methods:The adult worm soluble antigen of S. japonicum was purified and subjected to the two-dimensional electrophoresis and immunoblotting assay. The electrophoregram and immunoblotting pattern was obtained. Some bioinformation was obtained including molecular weight and isoionic point of the proteins with the PDQuest two-dimensional electrophoresis image analysis software and the spots of specific antigens were identified and among these 30 protein spots were picked up. LC-MS/MS was used for sequencing following trypsase digestion of the polypeptides in gel. Non-redundant protein sequence library of Schistosoma was searched with SEQUEST software. The peptide mass fingerprints (PMF) and sequencing data of 24 protein spots were successfully obtained, which belong to 18 proteins, for structural and functional analyses of these proteins by bioinformatics. Results:The soluble antigen of the S.japomicum adult worm were treated by two-dimensional electrophoresis,stainned by coomassie R250 .The electrophoretogram demonstrates about 152 major protein spots.The molecular weight is 14 kDa~134 kDa.And the isoelectric point mainly concentrate upon 4.94～9.50. assayed by Western blotting ,on the experimental group,the number of the blot which embody the reaction between the antigen and antibodies is about 57.While on the control group,the number of blot is zero. 30 protein spots were incised from coomassie staining gel and digested in gel by trypisin. 24 maps of PMF. were obtained and 18 proteins were identified. The PMF has high definition and powerful peak signal.It has more fragment peaks between m/z=200～2000. Analyze the sequence information of the polypeptide according to the acquried data and use the Data Base Retrieval(DBK) to determine the protein.By Internet on-line analysis and the soft of bioinformatics.At last,we got the information on the struture and function of protein. The result of the Data Base Retrieval demonstrate that the protein is correlation with cell movement or energy metabolism or signal transduction..It also has the functional correlation with the overlap,modification and synthesis of the protein.The C4 protein has the important biologic functional locus and domain similar to glyceraldehyde-3-phosphate dehydrogenase. Take further essay of the code geues,stucture and fuction of the C4 protein.The result of the sequence analysis show that the cDNA contain a open reading frame of 816 bp.It can encode 271 amino acid,the mole cule weight of the encode protein is 29.34 kDa,pI 9.12. The protein has the active site similar to the dehydrogenase of the glyceraldehyde phosphate,calmodulin binding dotain on N teminal,the catalytic domain retate to the transportation and metabolism of sugar and the potential epotope domain.Conclusion: Using the technique of two demengional electrophoress,mass sprctrum assay and bioinformatics to analyse the soluble antigen of the S.japomicun adult worm.Sucessfully assay several protein antigens of Sj AWA and demonstrate their structure and function.It provide new clue and ider for study on Sj AWA and setablish the base of the study on proteomics of the S.japonicum.It also provide the possiblity of getting the vaccines of the infection by schistosoma and the Diagnostic antigen of immunodignosis.