【摘要】 【目的】探索细粒棘球绦虫成虫表膜糖抗原的免疫性。【方法】细粒棘球绦虫成虫虫体冻融产物,经蛋白酶K消化,离心上清作为粗提表膜糖抗原,SDS-PAGE鉴定糖抗原成分,硫酸-蒽酮法测定多糖浓度,ELISA、Western-blot和IFA法分别检测免疫小鼠抗血清抗体效价和特异性以及确定抗原在虫体组织中的位置。【结果】SDS-PAGE图谱显示无蛋白条带出现,表明成虫膜蛋白被蛋白酶K完全降解;制备的糖抗原浓度为2.54mg.mL-1,初步确定为糖抗原。ELISA结果显示,随着免疫次数增加,抗血清中特异性抗体(IgG、IgM和IgA)效价也随之升高。与阴性血清相比,IgG、IgM和IgA的P/N值分别为4.9、3.2和6.4。未检测到特异性IgE。Wenstern-blot检测结果显示,抗血清与原头蚴粗提虫体蛋白、细粒棘球绦虫成虫表膜蛋白、表膜糖抗原有效识别。IFA显色结果显示糖抗原在细粒棘球绦虫虫体表膜中有表达。【结论】经蛋白酶K消化后提取的细粒棘球绦虫表膜糖抗原具有良好的的免疫原性和免疫反应性,有望用于包虫病的诊断和免疫。更多还原

【Abstract】 【Objective】 The immunological characteristics of the carbohydrate antigen(glycoprotein) of E.granulosus adult surface membrane were explored in this study.【Method】 The freez-thaw E.granulosus adult worm lysis was digested by protease K,and centrifuged to collect supernatant as crude glycoantigen materials,which was identified by SDS-PAGE,quantified by sulfuric acid anthrone method.Then,the Balb/c mice were injected hypodermically by the material six times for anti-serum,which was collected to measure immunoglobulin(IgG,IgM and IgA) by ELISA,and to determine the specificity by western-blot method.To localize the site of the antigen composition in the E.granulosus adult worm by IFA.【Result】 The SDS-PAGE showed that there was no visible band on the gel.This suggested that the proteins of the parasite surface membrane were degraded completely.The concentration of glycoantige was 2.54mg?mL-1 in the preparation.ELISA showed that the specific antibodies(IgG,IgM,and IgA) were rised gradually with the increase of the immunization times,and the positive/negative ration(P/N) of IgG,IgM and IgA equals to 4.9,3.2 and 6.4,respectively.Comparatively,the specific IgE was not detected.Western blotting showed that the E.granulosus adult worm surface membrane glycoantigen and native proteins could be recognized by the anti-serum.IFA proved that the glycoantigen was located on the surface membrane of E.granulosus adult worm.【Conclusion】 The glycoantigen of E.granulosus adult worms digested proteinase K has good immunogenicity and reactivity,and could be used to develop vaccine candidates and diagnosis material.更多还原