Screening And Application Of Epitopes Derived From Serpin And P46 Proteins Of Trichinella Spiralis

Trichinellosis is a food-borne zoonotic parasitic disease caused by Trichinella spiralis,which is widely distributed throughout the world.T.spiralis has a wide range of hosts and can infect humans and more than 150 animals.Humans are infected mainly by eating raw or semi-raw meat or meat products containing muscle larvae of Trichinella spiralis.Humans infected with Trichinella spiralis have no obvious clinical symptoms,and clinical diagnosis is difficult.Severe patients may die of complications from trichinellosis if they delay to diagnose and are not treated promptly.Trichinellosis not only brings huge economic loss to animal husbandry,but also causes serious harm to human health.ELISA is the prior immunological method for diagnosis of trichinellosis using T.spiralis muscle larval ES antigens to detect anti-Trichinella antibody Ig G.However,the method usually leads to false negative results due to the existence of"window period".Meanwhile,ES antigens could cross-react with sera infected with other parasites.Therefore,it is necessary to screen novel diagnostic antigens for porcine trichinellosis,which are easy to be prepared in large quantities,with high sensitivity and good specificity.In this study,the Serpin protein of Trichinella spiralis was firstly expressed in segments,and the reactivity of each segment protein was verified by western blot.Then the antigen epitope of Serpin protein were analyzed by Jameson-Wolf module in DNAStar.The results from both antigen epitope analysis and western blot analysis were used to predict the peptide sequences with strong reactivity.The sequences were designed into different short peptides,and the reactivity of each short peptide was verified by dot hybridization.The epitopes with strong reactivity were synthesized in tandem,and their reactivity was evaluated by dot hybridization and indirect ELISA.The western blot results showed that the Ser2 and Ser5 fragments derived from Serpin protein could react with the sera from pigs infected with T.spiralis.Nine candidate short epitopes were designed from the sequence of S₁₄₉-Y₂₀₅,which were named as S₁₄₉-A,A₁₅₄-A,I₁₅₉-V,I₁₆₄-H,S₁₆₉-K,F₁₇₄-P,T₁₇₉-K,F₁₈₄-T and S₁₉₀-Y in order.The reactivity of the candidate short peptides were verified by dot hybridization,and two of them,F₁₇₄-P and F₁₈₄-T,had strong reactivity.The Serpin EFP-63 consists of F₁₇₄-P and F₁₈₄-T,which had good reactivity verified by dot hybridization.The indirect ELISA method established by Serpin EFP-63 antigen had no cross-reaction with the sera of pig infected by T.gondii and C.cellulosae.The coincidence rates of positive sera and negative sera were 72.7%and 94.7%,respectively,compared with those detected by ES antigen.The Escherichia coli BL21（DE3）/p ET30a-P46 preserved in our laboratory was induced and expressed to obtain P46 recombinant protein.Western blot and indirect ELISA were used to verify the reactivity of P46 recombinant protein.The Jameson-Wolf module in DNAStar was used to screen and verify the main epitopes of P46 protein.Western blot results showed that the P46 recombinant protein had good reactivity.The indirect ELISA method established by the r P46 protein had no cross-reaction with sera from pig infected by T.gondii and C.cellulosae.The coincidence rates of positive serum and negative serum were 27.2%and 100%,respectively,compared with those detected by ES antigen.The results showed that the detection effect of P46 recombinant protein was lower than that of ES antigen.However,the established indirect ELISA method again proved the reactivity of P46 recombinant protein,indicating that P46 recombinant protein can be used for the screening of subsequent polypeptide antigens.A total of 9 candidate short peptides were obtained by antigen epitope analysis,but only 2short peptides had strong reactivity through dot hybridization,whose sequences were D₂₈₇-M and S₃₇₃-C.In this study,the epitope peptipes were successfully identified as strong reactivity dervied from Serpin and P46 proteins of Trichinella spiralis.An indirect ELISA method was developed using Serpin EFP-63 antigen,which was concatenated by the epitopes of Serpin protein.The present study could provide candidate the candidate antigens for the diagnosis of porcine trichinellosis.