Serological Screening And Identification Of Specific Diagnostic Epitopes Of Echinococcus Granulosus Protoscolex

Objective To screen the peptide molecules with diagnostic potential in the specific expressed antigens of protoscolex by bioinformatics analysis,so as to lay a foundation for the development of specific diagnostic antigens of echinococcosis.Methods 1.Differential gene expression profile analysis was used to screen the specifically expressed molecules of Echinococcus granulosus protoscoles:(1)through the comparative analysis of the gene expression profiles of Echinococcus granulosus protoscoles and six hooked cercariae published by the National Southern Genome Research Center of China on the Internet NCBI website genome database,the differentially expressed genes were screened out.According to the saturation value(Reads)and expression abundance(RPKM),the differential molecules that were not expressed(Reads=0,RPKM=0)in the stage of cercariae,but highly expressed in the stage of protoscolex(Reads > 20,RPKM > 0)were screened.(2)using the literature notes of Uniprot online protein database,the subcellular localization of the selected differential molecules was analyzed,and the expressed or secretory proteins on the membrane surface were screened as candidates for diagnostic antigens.2.The preparation of serum model: the serum of mice in blank group was not treated,the serum of mice in adjuvant control group was immunized with Freund'sadjuvant,and the serum of mice in protoscolex infection group was obtained by directly infecting mice with proto-cercariae.The sera of protoscolex crude antigen immunized group were immunized with protoscolex samples.Mouse serum of candidate molecular protein immunization group: the genetically engineered strain of candidate molecule was constructed and immunized mice after expressing its recombinant protein.3.Based on the above serum model,Analysis of immune characteristics of candidate molecules: the immunological characteristics of diagnostic antigen candidate molecules were identified by Western-blot method,and the serum recognition effect of candidate molecules was analyzed by ELISA method.4.Bioinformatics prediction analysis of differential molecular epitopes and synthesis and identification of epitope peptides:(1)B cell epitope antigens of candidate molecules were cross-analyzed and screened by online prediction and analysis websites ABCpred,IEDB and BepiPred-1.0,and the secondary structure of candidate molecules was comprehensively predicted and analyzed by DNAstar software,and multiple epitope peptides with antigen potential were obtained by combining the above biological information.(2)the selected epitope peptides were artificially synthesized by Shengong Bioengineering(Shanghai)Co.,Ltd.,and the quality of the synthetic peptides was identified by high performance liquid chromatography(HPLC)and mass spectrometry(MS).5.Analysis and screening of immunological characteristics of epitope peptides: ELISA method was used to identify all the synthesized epitope peptides with different serum models,and the peptides that could recognize both protoscolex infection group and candidate differential molecular recombinant protein immune group were analyzed and screened.In addition,the serum specificity of the selected epitope peptides was verified.Results 1.Through the comparative analysis of the mRNA molecular expression profiles of Echinococcus granulosus protoscolaria and six hook cercariae,7825 differential molecules were screened out which were not expressed in the six hook cercariae stage,while a total of 356 molecules were highly expressed in the protoscolex stage.After subcellular localization analysis,16 expressed proteins on the membrane surface or secreted proteins were found,and 5 molecules with too long or too short sequence length were removed,and finally 11 candidate molecules were left.Eg-07112 and Eg-07279,were selected as the final candidate diagnostic antigens by random number table method.2.The prepared serum model was used to identify the candidate molecules.Western-blot results showed that the recombinant protein rEg-07112 could not recognize the sera of mice in blank group and adjuvant control group,but could be recognized in sera of mice immunized with crude antigen of protoscolex,protoscolex infection group and rEg-07112 protein group.The recombinant protein rEg-07279 could not recognize the sera of mice in blank group and adjuvant control group,but could be recognized in sera of mice immunized with crude antigen of protoscolex,protoscolex infection group and rEg-07279 protein group.The results of ELISA showed that both rEg-07112 and rEg-07112 could recognize the sera of mice infected with protoscolex,the sera of mice immunized with crude antigen of protoscolex and the sera of mice immunized with intact protein,and the recognition ability of mice immunized with intact protein,the sera of mice infected with protoscolex and the serum immunized with crude antigen of protoscolex were in the order of strong to weak.3.The amino acid sequences of Eg-07112 and Eg-07279 proteins were comprehensively analyzed by B cell epitope online prediction websites ABCpred,IEDB,BepiPred-1.0 and DNAstar software.Finally,9 peptides of Eg-07112 epitopes and 9 peptides of Eg-07279 epitopes were obtained,with a total of 18 peptides.The purity of the synthesized peptides was analyzed by high performance liquid chromatography(HPLC).The results showed that the purity of the peptides was more than 98%(98.030% Mel 99.556%).The molecular weights of synthetic peptides were identified by mass spectrometry(MS).The results showed that the molecular weights of synthetic peptides were consistent with those expected,indicating that the quality of synthetic peptides was stable and reliable.4.The serum model of synthetic epitope peptides was identified by ELISA method.When the S/Co of epitope peptides in protoscolex infection group and Eg07112 and Eg07279 protein immunization group were set to ? 2.0,two peptides were screened,the specific sequences were Eg-07112(275-PNPWAPPQQQTETT-288)and Eg-07279(468-SDASKKPATYNPDAYP-483),which showed good specificity in a small range of serum model.Conclusion 1.The differential molecules Eg-07112 and Eg07279 screened by bioinformatics analysis have the potential to become specific diagnostic antigens of Echinococcus granulosus.2.Antigenic epitopes Eg-07112(275-PNPWAPPQQQTETT-288)and Eg-07279(468-SDASKKPATYNPDAYP-483)have the potential to become specific diagnostic antigen molecules of Echinococcus granulosus,and the recognition advantage of serum is stronger than that of intact protein molecules.