Study On Similar Research Of Common Components Between Schistosoma Japonicum Dissovle And Whitmania Pigra Heat Secretions

AimNowadays, Schistosomiasis, which is a serious illness harmful to human health, was still widespread in our country along the area of the yangtze river, such as Hunan, Hubei, Jiangxi, Anhui and Jiangsu etc.Currently the immunology diagnosis technique is mainly the important component parts of serum immunodiagnosis. However, because of the complexity diversity of antigens in Schistosoma during the whole life cycle, cross-reactions are very common in immunoreactions.As documents described that the common mimic epitopes between Lumbricus terrestris(Lt) and Schistosoma japonica(Sj) sibling annelid. The similar molecular weight with immunologically competent common compositions between Schistosoma japonica and Lumbricus terrestris heat secretions maybe enzyme, such as protein kinase.Qin Zhiqiang reported that much more antibody against Leech soluble antigen in sera of patients with Schistosomiasis. Result of Leech soluble antigen ELISA showed high sensitivity and specificity in diagnosing schistosomiasis japonica.Whitmania pigra is attached to Hirudinea of Annelida, which have 400-500 species in the world, but there is no report about the common antigen between Schistosoma Japonicum dissolvable(Sj-Ds) and Whitmania pigra heat secretions(Wp-hs).Therefore, Wp is worthwhile to study like Lt as a special antigen in the diagnostic research of schistosomiasis.To explore the molecular mechanism of immune cross-reactions between Sj-Ds and Wp-hs, Dot-ELISA,SDS-PAGE and Western blot was used to analyze the cross reactivity between them. Then PDT was to obtain the common mimic epitopes between Wp-hs, Sj and Lu, and to comparison the amino acid sequence similarity with biology informatics mothod for them. Then SDS-PAGE and Western blot were applied to study the cross-rectivity of the common mimic epitopes. We hope the results of this study could provide theoretical and experimental basis for the research on immune cross-reactive phenomena among these parasitic helminthes.Methods1. Preparation and cross-reactivity analysis of Sj-Ds and Wp-hs(1) Preparation of heat secretions from WpThe Stress proteins from Wp were acquired by hyperthermia stimulation with 40℃for 30 minutes.(2) Preparation of dissolvable from SjThe dissolvable from Sj were acquired through grinded by tissue homogenizer.(3) Cross-reactivity analysis between Sj-Ds and Wp-hsSDS-PAGE was performed to isolate the protein components of the Sj-Ds and Wp-hs. Western blot was used to analyse the cross-reactivity of the Sj-Ds and Wp-hs with sera from schistosomiasis patients and healthy people.2. Screening and cross-reactivity analysis of common mimic epitopes between Sj,Lu and Wp-hs.(1) Screening of common mimic epitopes between Sj,Lu and Wp-hs by PDT.The phage 12-mer peptide library was screened by Wp-hs-Ig, Lu-Ig and Sj-Ig,or Wp-hs-Ig, Sj-Ig and Lu-Ig in turn. After two rounds of panning, 20 positive plaques were selected from each plate respectively. The antigenicity of each clone was examined by ELISA, and clones which had good immunocompetence were sequenced.(2) Cross-reactivity analysis of common mimic epitopes.SDS-PAGE was performed to isolate the protein components of the common mimic epitopes between Sj,Lu and Wp-hs. Western blot was used to analyse the cross-reactivity of the mimic epitopes with sera from schistosomiasis patients.3. Bioinformatics analysis about amino acid sequence of mimic epipopesApply Blast software to comparison the sequence similarity about the common mimic epitopes of Sj,Lu and Wp-hs,schistosoma,paragonimus westermani,Trichinella spiralis between schistosoma protein data bank and lumbricus terrestris protein data bank in SWISS-PROT protein data bank.Results1. Preparation and cross-reactivity analysis of Sj-Ds and Wp-hs.There were many similar molecular weight (12.6 - 85.4 kDa) protein bands between Wp-hs and Sj-Ds, and some protein bands between 21.6 - 95.4 kDa from Wp-hs had intensive cross reaction with the sera of patients with schistosomiasis, but no cross reaction with the sera of healthy people. Some components of Sj-Ds whose molecular weight was from 40.1 kDa to 89.0 kDa could combine with the sera of rabbits immunized with Wp-hs. There are at least three mutual immunocompetent protein bands with similar molecular weight between W. pigra and S. japonicum.2. Screening and cross-reactivity analysis of common mimic epitopes between Sj,Lu and Wp-hs. (1) Screening of common mimic epitopes between Sj,Lu and Wp-hs by PDT.After two rounds of panning, three positive clones were acquered and sequenced. Then they were translated and analysed by Blast software. The results showed that there were the same discontinuous amino acids sequences (LP-AI) between E-Sj and actin in lumbricus terrestris and schistosoma; as the same uncontinous amino acids sequences (SSL) between Sj-E and eggshell protein 2A precursor of Sj, and cytochrome coxidase subunit 3 of Lt; as the same continous amino acids sequences (TRLR) between Wp-hs and HMG-CoA reductase of Sj; 60S ribosomal protein L13 of Lt.(2) Cross-reactivity analysis of common mimic epitopes.Sera from schistosomiasis patients were used to study the cross-reactivity of Wp-hs,E-Sj,Sj-E by Western blot, and the results showed that there were all positive bands at 65.4 kDa.2. Bioinformatics analysis about amino acid sequence of mimic epipopesThe results showed that there were the same continous amino acids sequences (LPGA) between Sj1 and heat shock 70kDa protein of Sj, NADH dehydrogenase subunit 5 of Lt; as the same uncontinous amino acids sequences (HY-KS) between Pw6 and E3 ubiquitin-protein ligase of Sj, NADH dehydrigenase subunit 5 of Lt; as the same uncontinous amino acids sequences (LS-PL) between Ts4 and 6-phosphofructokinase of Sj; NADH dehydrogenase subunit 5.Conclusions1. There are at least three mutual immunocompetent protein bands with similar molecular weight between W. pigra and S. japonicum, which illustralate the phenomenon of immunocompetent cross-reactionSj-Ds and Wp-hs.2. The same amino acid sequence could be found with PDT in protein data banks between Sj and Lt, which further confirm that Sj and wp-hs have the common immunocompetent antigen. The common antigen is whether or not stress protein, it need to be further studed.3. Common mimic epipopes between Sj and Lu could be acquired by screening phage with sera from schistosomiasis patients and rabbit immunized with Lu.4. The common mimic epitopes between schistosoma,paragonimus westermani,Trichinella spiralis could be acquired by screening phage, which the same amino acid sequence could be found in protein data banks between Sj and Lt. the results confirmed that the substance is a highly conservatived protein in evolutionary process, and it may be some kind of protein kinase.