| Objective By using proteomics technology to screen of Echinococcus granulosus protoscolices and cystic wall,which is expected to become the molecular immunology diagnostic antigen,providing a theoretical basis for the early diagnosis of hydatid disease.Methods We extract the protein of protoscolex,cystic wall of hydatid cyst,using SDS-PAGE and Western blot technology,to preliminarily understand the protein expression patterns and immunogenicity of protoscolices and cyst wall;The protoscolices and cyst wall proteins were separated by two-dimensional electrophoresis.we select 40 protein spots in protoscolices and 8 protein spots in cystic wall,then enzymatic treatment by trypsin.Using mass spectrometry technology to obtain the protein peptide fingerprinting data,and searching NCBInr and SWISS-PROT protein database to identify the protein spots by Mascot software;Through consulting literature and using bioinformatics technology,enolase and transketolase been selected which has identified by mass spectrometry,may become candidate antigen molecules.Then using protparam,protscale,signalp,sompa of expasy system and Sub Locv1.0,DNAStar,IEDB,SYPEITHI,Phyre2 software,etc,to further analyze the physicochemical properties,B/T cell epitopes,transmembrane domains,subcellular localization,secondary structure and tertiary structure of enolase and transketolase and evaluate the possibility to become specific diagnosis antigen.Construction of enolase,transketolase prokaryotic expression vector,access to purified protein for later experiments to provide a basis.Results 1.The protoscolex protein concentration in the molecular weight of about 72 k D,the cyst wall protein concentration in 72 k D,26 k D and 17 k D,DAB show colois very sensitive,which hint that antigen molecules with strong immunogenicity exist in the protoscolex and cyst wall protein.2.There are 36 and 6 proteins been identified in the protoscolex and cyst wall,respectively,which can be roughly divided into:(1)the cytoskeletal proteins(2)enzymes related to metabolism(3)cell signal transduction proteins(4)transport related proteins(5)heat shock proteins(6)protein translation related proteins.3.Full-length enolase gene is 1449 bp,composed with 3exons and 2 introns,the CDS is 1302 bp,encoding 434 amino acids;the relative molecular weight is 46.56 k D and isoelectric point is 6.48,has a long half-life,instability index is 32.97,belong to stable protein.The hydrophilic,flexible regions,antigenicity,surface accessibility scores amino acid regions of enolase were 9,12,19,8,respectively;the possible B cell linear epitopes is15;enolase protein may has 10 CTL cell epitopes and 10 Th cell epitopes.In the secondary structure,alpha helix and irregular coil.The amino acid sequences has 66.23% homology with human enolase.Transketolase gene consists of 7 exons and 6 introns,the CDS is 1878 bp,encoding 625 amino acids;The relative molecular weight is 67.76 k D;The prediction results show that transketolase is transmembrane protein located in the cytoplasm,mainly in the secondary structure is alpha helix and random coil;Transketolase may has 16 B linear potential cell epitopes,11 CTL cell epitopes and 13 Th cell epitopes;The amino acid sequences hasonly 58.68% homology with human transketolase.4.The prokaryotic expression vector of pet28a-Eg EN and pet28-Eg TK was successfully constructed and the recombinant protein was obtained.The positive rate of serum reaction of Eg En,Eg TK and CE patients was greater than 75%,and CE and AE had partial cross reactivity.Conclusion Some protein spots in protoscolex and cyst wall of Echinococcus granulosus has been preliminarily identified,involved into cytoskeleton,metabolism,signal transduction and protein synthesis.Preliminary build the protoscoleces and cystic wall protein expression database of Echinococcus granulosus in Qinghai province.Enolase,transketolase of Echinococcus granulosus may become specific antigen in early diagnosis,but need further experiments to confirm. |
PDF Full Text Request