Development Of A Detection Method For Trichinella In Swine Based On Serine Protease Inhibitor WM5

Trichinellosis is a severe zoonosis caused by eating raw or semi-raw meat containing Trichinella spp.It not only seriously threatens public health,but also causes significant economic losses to the husbandry industry and meat export.At present,indirect ELISA method based on excretory/secretory?ES?products of muscle larvae?ML?is the only serological inspection method recommended by Office international des Epizooties?OIE?.Because the preparation of ES antigen is difficult,development of a simple,convenient,rapid and specific method for detection of trichinellosis has great importance to protect public health,international meat trade and reduce the economic loss of animal husbandry.Serine proteinase inhibitor?Serpin?is a protein superfamily with conserved structure and can inhibit serine proteases from playing important physiological roles in various biological systems such as coagulation,complement activation and inflammation.At present,serine protease inhibitors have been proved to transcribe at different developmental stages of Trichinella spiralis?T.spiralis?,but there has been no systematic study on construction of detection method based on serine protease inhibitor WM5 from T.spiralis.In the early detection of our laboratory,the highly abundant and reactive serine protease inhibitor WM5 was obtained from the T.spiralis ML cDNA library.In this study,we expression and purification recombinant WM5?rWM5?protein by using prokaryotic expression system.The identification results indicate that rWM5 could be specifically recognized by swine serum infected with T.spiralis,so rWM5 might be a good candidate antigen for detection of T.spiralis.By preparation and identification of polyclonal antibody,the titer of the polyclonal antibodies is 1:160 000.The results of Western blot showed that the prepared polyclonal antibodies could specifically recognize crude worm extract,ML ES and rWM5,suggesting that WM5 might be a secretory protein.An indirect ELISA method based on rWM5 was established.After optimizing the conditions of each step of the reaction,identification of 205 pigs without trichinellosis confirmed that the cut-off value of the established indirect ELISA method was 0.207,and there was no cross-reaction with other parasite-positive sera.The intra-and inter-assay coefficients of variation are less than 10%.Compared with the ELISA method based on ML ES antigen,the detection effect was similar.This method avoids the difficulties in preparing ML ES antigen of T.spiralis.Therefore,the purified rWM5 is more suitable for industrial production and large-scale detection of T.spiralis in swine.Finally,we produced monoclonal antibodies from rWM5 immunized BALB/c mice,and obtained two monoclonal hybridoma cell lines named 2B3 and 3F11,respectively.The subtypes of these monoclonal antibodies were identified to IgG1;the Western blot analysis indicated that the monoclonal antibodies could recognize WM5 in ML ES and crude worm extract.Localization of WM5 was performed with worm bodies of T.spiralis at different developmental stages using immunofluorescence assay.Signals were observed in the stichosome of the ML and the epidermis of the adult worm and ML,suggesting that WM5 was produced from the stichosome.The epitopes of the two monoclonal antibodies were identified on the²⁷⁷EALKKLGIEDLF²⁸⁸ amino acid sequence.Monoclonal and polyclonal antibody preparation have a great significance for the detection of Trichinella infection and the establishment of serological detection methods.