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目的采用免疫共沉淀偶联质谱技术分离和鉴定旋毛形线虫肌幼虫代谢分泌抗原中的血清学抗原。方法收集感染旋毛形线虫的新西兰兔血清,硫酸铵沉淀法分离血清Ig G;从感染肌肉中分离纯化肌幼虫并培养收集代谢分泌抗原。免疫共沉淀和SDS-PAGE分离代谢分泌抗原,Western blot法分析血清学抗原,并予质谱鉴定。结果间接ELISA检测旋毛形线虫感染新西兰兔的血清抗体滴度达1∶6400以上;采用硫酸铵沉淀法获得血清Ig G。免疫共沉淀的旋毛形线虫肌幼虫代谢分泌抗原经电泳分离获得4条较清晰的蛋白质条带,Western blot法分析发现在相对分子质量(Mr)40 000、50 000、83 000处存在旋毛形线虫感染血清识别、而正常兔血清不识别的3条较强的蛋白质条带。切取Mr40 000、50 000、83 000处的蛋白质条带进行质谱鉴定,获得4种蛋白质分子,分别为丝氨酸蛋白酶、肌幼虫特异性丝氨酸蛋白酶、43 000分泌糖蛋白、53 000代谢分泌抗原。结论采用免疫共沉淀偶联质谱技术从旋毛形线虫肌幼虫代谢分泌抗原(ES)中获得4种血清学抗原,为旋毛形线虫病有效诊断和疫苗候选分子的获得提供了新的来源和视角。
OBJECTIVE: To isolate and identify serological antigens in the secretion and secretion of A. niger larvae by immunoprecipitation coupled with mass spectrometry. Methods New Zealand rabbits infected with Trichinella spiralis were collected and serum Ig G was separated by ammonium sulfate precipitation. Muscle larvae were isolated and purified from infected muscles and cultured for collection of secreted and secreted antigens. Immunoprecipitation and SDS-PAGE were used to separate and secrete antigens, Western blot was used to analyze the serological antigens and identified by mass spectrometry. Results Indirect ELISA showed that serum antibody titers of New Zealand rabbits infected with Trichinella spiralis were up to 1:6400. Serum Ig was obtained by ammonium sulfate precipitation. The immunoprecipitated A. californicus larvae metabolized and secreted antigens were separated by electrophoresis to obtain 4 clearer protein bands. Western blot analysis revealed the presence of Trichinella spiralis at relative molecular mass (Mr) of 40 000, 50 000 and 83 000 Serum was identified by infection, whereas normal rabbit serum did not recognize 3 strong protein bands. The protein bands of Mr40 000, 50 000, and 83 000 were cut out for mass spectrometry identification. Four protein molecules were obtained, namely serine protease, muscle larvae-specific serine protease, 43 000 secretory glycoprotein and 53 000 metabolic secretory antigen. CONCLUSION: Four serological antigens were obtained from the metabolic secretion antigens (ESs) of Myasthenia gracilis muscle by immunoprecipitation coupled with mass spectrometry, which provided a new source and perspective for the effective diagnosis of diapause nematodes and the acquisition of vaccine candidate molecules.