Separation,purification And Immunological Identification Of Soluble Antigen Of Echinococcus Multilocularis

Objective This paper is mainly about the separation and purification of Protoscolex soluble protein of Echinococcus multilocularis and enrichment of micro-components of protein,all the components underwent a further immunological identification,to predict the secondary structure and the Antigenic epitope for the Target Protein antigen in order to reveal the dominant epitopes of the antigen.Methods 1.Through the use of HiTrap Q FF and Mono Q 5/50 GL anion exchange column,the Protoscolex soluble antigen of Echinococcus multilocularis was separated into multiple different components,which were then put together with the serum of patients with multilocular hydatid cyst and the patients with echinococcosis granulosis cyst and that of healthy subjects for an immunoblotting and mass sepcturm identification,followed by a further BLAST aligning in order to obtain a highly specific antigen of multilocular hydatid cyst.2.The secondary structure of the protein was analyzed using SOPMA server.The T-cell and B-cell epitopes of Em LAP were predicted using IEDB,Syfpeithi,Bcepred and ABCpred online software.Results 1.A total of 9 components was obtained by the enrichment of HiTrap Q FF and Mono Q 5/50 GL anion exchange column,the result of further Western-blot showed that,the interest protein having a good immunogenicity and relatively high specificity was located at 55 kDa,having being identified by mass spectrum,20 proteins(Top 20)were screened out as the result of identification of interest protein according to origin of species,scoring,coverage rate of amino acid,total amino acid count and protein molecular mass,the Top20 proteins underwent a BLAST aligning,of which the result had been analyzed,wherein,the Echinococcus multilocularis-derived leucyl aminopeptidase(LAP)was the most different from the echinococcus granulosus-derived amino acid sequence,the similarity between both was 62.63%.2.The Alpha helix accounted for 45.42%,the Extended strand accounted for 16.57%,the Beta turnaccounted for 8.58% and the Random coil accounted for 29.43% of the secondary structure of the EmLAP,respectively.Following hioinformatic analysis,numerous distinct antigenic epitopes of EmLAP were identified.The high-scoring T-cell epitopes were located at positions L65-73,L75-84,L174-186,L233-240,L301-308,L333-343,L392-400,L445-454,L482-490;whilst the likely B-cell epitopes were located at positions L13-22,L39-47,L75-84,L98-110,L146-154,L174-186,L183-191,L233-240,L247-254,L281-288,L295-306,L319-328,L330-337,L339-347,L392-400,L397-410,L421-428,L464-473,L493-504,L504-512.Conclusion 1.There were not found other human-derived and parasite-derived proteins with a similar sequence in Echinococcus multilocularis-derived leucyl aminopeptidase,which means a relatively high specificity and can be regarded as a candidate molecule for the immunological diagnosis of Echinococcus multilocularis.2.Nine T-cell and twenty B-cell dominant epitopes of the EmLAP antigen were revealed by the hioinformatic methods which may he useful for the development of a dominant epitope vaccine.