Study On Molecular Mechanism Of Immune Cross-reaction Between Lumbricus Terrestris And Schistosoma Japonicum

AimThe accuracy of immunology detection is mainly determined by the properties of diagnostic antigens. Currently the diagnostic antigens of schistsomiasis japonica mainly include the AWA(adult worm antigens) and SEA(soluble egg antigens), both of which are rude and composed of complicated components, cross-reactions are very common among the clinic parasitic helminthes.In order to reduce the cross-reaction rate appeared in the diagnosis, many scholars have devoted themselves to searching for specific and sensitive diagnostic antigens, but few people have studied the mechanism of cross- reactions.Wisnewski reported in 1995 that the antigens of Lumbricus terrestris(Lt) could bind to the sera from schistosomiasis patients, but couldn't react with the sera from healthy persons; The inhibitory rate of competitive ELISA is above 30%; The mice vaccinated with LtAg could induce 36% protection against Schistosoma japonicum (Sj ) infection, and the antibodies raised by Lt could bind to several components of AWA.We have researched the cross-reactions between Lt and Sj, or Paragonimus Westermani(Pw), or Taenia solium(Tc), or Trichinella spiralis(Ts) many years, and found that LtAg could decrease the cross-reactive rates among these parasitic helminthes; There is no kind of antigen which can band to the serum from schistosomiasis, paragonimiasis, cysticercosis and trichinosis patients, and can't react with the serum from healthy person but LtAg .Therefore, LtAg is worthwhile to study as a special antigen in the diagnostic resea- rch of schistosomiasis. To explore the molecular mechanism of immune cross-reactions between Lt and Sj, PDT was used to obtain the common mimic epitopes between Lt and Sj, and grads salting out method was used to obtain the lumbricus soluble proteins. Then SDS-PAGE and Western-blot were applied respectively to study the the molecular components and cross-reactivity of the common mimic epitopes, depositions, lumbrukinase and stress proteins from Lt, 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. Screening and cross-reactivity analysis of common mimic epitopes between Lt and Sj.(1) Screening of common mimic epitopes between Lt and Sj by PDT.The phage 12-mer peptide library was screened by Lt-Ig and Sj-Ig, or Sj-Ig and Lt-Ig in turn. After two rounds of panning, 21 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 epitopesSDS-PAGE was performed to isolate the protein components of the common mimic epitopes between Lt and Sj. Western-blot was used to analyse the cross-reactivity of the mimic epitopes with sera from schistosomiasis patients.2. Preparation and cross-reactivity analysis of depositions from Lt by grads salting out(1) Preparation of depositions from Lt by grads salting outThe Lumbricus soluble proteins were deposited by grads salting out with 30%,40%,50%,60%,70%(NH4)2SO4 in turn. (2) Cross-reactivity analysis of the depositionsSDS-PAGE was performed to isolate the protein components of the depositions. Western-blot was used to analyse the cross-reactivity of the depositions with sera from schistosomiasis patients.3. Cross-reactivity analysis of lumbrukinaseThe methods are the same as the cross-reactivity analysis of the depositions from Lt by salting out.4. Preparation and cross-reactivity analysis of stress proteins from Lt(1) Preparation of stress proteins from LtThe stress proteins from Lt were acquired by hyperthermia stimulation with 40℃for 30 minutes.(2) Cross-reactivity analysis of the stress proteinsThe methods are the same as the cross-reactivity analysis of the depositions from Lt by salting out.Results1. Screening and cross-reactivity analysis of the common mimic epitopes between Lt and Sj.(1) Screening of common mimic epitopes between Lt and SjAfter two rounds of panning, six positive clones were acquired and sequenced. Then they were translated and analysed by Blast software. The results showed that there were the same continous amino acids sequences(LAET) between Lt9 and gynecophoral canal protein of Sj, NADH dehydrogenase subunit 1 of Lt ; as the same uncontinous amino acids sequences(HT-HI) between Sj17 and glutamine synthetase of Sj, elongation factor 1-alpha of Lt; as the same continous amino acids sequences(NSIL) between SjLt11 and lactate dehydrogenase-like protein of Sj, NADH dehydrogenase subunit 6 of Lt.(2) The cross-reactivity analysis of Lt9,Sj17,SjLt11Sera from schistosomiasis patients were used to study the cross-reactivity of Lt9,Sj17,SjLt11 by Western-blot, and the results showed that there were all positive bands at 64.9 kDa .2. The SDS-PAGE and Western-blot results of depositions from Lt by grads salting outMany protein bands with MW of 25.3～112.7kDa and <18.3kDa could be acquired by 30%～70% (NH4)2SO4 grads salting out. The reactive bands with MW of 20.4～110.2kDa were observed after adding the sera from schistosomiasis patients.3. The SDS-PAGE and Western-blot results of lumbrukinaseThe reactive bands with MW of 39.8～95.1kDa were observed by adding the sera from schistosomiasis patients to the protein bands with MW of 26.7～97.8kDa identified by SDS-PAGE.4. The SDS-PAGE and Western-blot results of stress proteins from Lt.The reactive bands with MW of 61.2～95.1kDa were observed by adding the sera from schistosomiasis patients to the protein bands with MW of 28.3～97.8kDa and <19.6 kDa identified by SDS-PAGE.Conclusions1. Three common mimic epitopes between Lt and Sj could be acquired by screening phage with sera from schistosomiasis patients and rabbit immuned with Lt.2. Grads salting out with saturate ammonium sulphate could extract the lumbricus soluble proteins effectively, which had cross-reactive antigens with MW of 20.4～ 110.2kDa between these soluble proteins and Sj.3. There were cross-reactive antigens with MW of 39.8～95.1kDa between lumbrukinase and Sj.4. There were cross-reactive antigens with MW of 61.2～95.1kDa between stress proteins from Lt and Sj.The common mimic epitopes between Lt and Sj, depositions, lumbrukinase and stress prtiens from Lt all had the cross-reactive bands with MW of 61.2～95.1 kDa recognized by the sera from schistosomiasis patients. The common molecular components with MW of 61.2～95.1kDa between Lt and Sj may be some enzymes and have NSIL, LAET and HT-HI sequences. These components play an important part in the immune cross-reaction between Lt and Sj, and perhaps are the important molecular basis of immune cross-reaction among the parasitic helminthiasis.In a word, the molecular components of cross-reactions between Lt and Sj may be some enzymes, lumbricus soluble proteins as special antigens have spacious prospect in diagnosis of parasitosis.