采用分子筛层析法分离纯化细粒棘球蚴囊液粗抗原  

Purification of antigens from hydatid cyst fluid by molecular sieve chromatography

作　　者：张颋[1] 武洁雯 王冬[2] 高海军[3] 叶萍[3] 陈英[1] 莫筱瑾[1] 徐斌[1] 冯宇[2] 

机构地区：[1]中国疾病预防控制中心寄生虫病预防控制所,卫生部寄生虫病原与媒介生物学重点实验室,世界卫生组织疟疾、血吸虫病和丝虫病合作中心,上海200025 [2]甘肃省疾病预防控制中心,兰州730020 [3]四川省甘孜州疾病预防控制中心,康定626000

出　　处：《国际医学寄生虫病杂志》2015年第3期132-136,共5页International JOurnal of Medical Parasitic Diseases

基　　金：国家科技重大专项(2012ZX10004220);国家自然科学基金(81201315);中国博士后基金(2014M551331);甘肃省科技支撑计划(1304FKCA120);卫生部病原与媒介重点实验室开放课题(WSBKTKT201304);甘孜州青年科技人才培养计划(2012203)~~

摘　　要：目的 分离纯化细粒棘球蚴囊液抗原，去除其中的非特异性反应蛋白，探索优化囊液抗原制备方法。 方法 采用超滤法浓缩羊肝细粒棘球蚴囊液制备成粗抗原，并采用免疫印迹实验对囊液抗原进行分析，发现非特异性反应的主要蛋白条带。随后采用Superdex凝胶柱通过分子筛层析法对囊液抗原进行纯化。用44份细粒棘球蚴病患者、17份健康人和10份囊尾蚴患者血清进行酶联免疫实验，评估纯化后囊液抗原的检测能力。 结果 免疫印迹实验发现，非特异性反应的主要条带的相对分子质量（Mr）约为55 000，纯化后去除了该非特异性反应蛋白。纯化后的囊液抗原ELISA检测灵敏度为93.2%（41/44），特异性为94.1%（16/17），约登指数为0.87，与囊尾蚴病交叉反应性为30%（3/10）。未纯化前的灵敏度为90.9%（40/44），特异性为88.2%（15/17），约登指数0.79，与囊尾蚴病交叉反应性为60%（6/10）。纯化前后ELISA检测的灵敏度存在显著性差异。 结论 本研究采用分子筛层析法对羊源细粒棘球蚴的囊液抗原进行纯化，去除了引起非特异性反应的主要蛋白（Mr 55 000），为后续进行囊液抗原标准化、提高检测能力提供了依据。To optimize the method for preparation of h ydgtid eyst fluid antigen by purifying crude antigen to remove nonspecific reactive proteins. Methods Curde antigen was obtained from sheep hydatid fluid by ultra filtration, and analyzed by Western blotting to identify nonspecific immunorsantive bands. For purification, molecular sieve chromatography with Superdex GL selumn was used to remove primary nonspecific reactive proteins. The purified antigen was evaluated for diagnostin efficacy using immunoblotting and ELISA with the sera of 44 cystic eohinococcosis （CE） patients, 17 healthy subjects and 10 cysticercusis patlanis, Realuts A primary nonspecific proteins band of Mr 55 000 was identified by Western blotting probed with inhered and normal sets. After purification, immunoblotting domenstrated that the nonspecific component was removed from the crude antigen successfully. The sensitivity, specificity, Yourdon＇s index, and cross-reaction with cysticercosis of the purified antigen in ELISA were 93.2% （41/44）, 94.1% （16/17）, 0.87 and 30%（3/10） respectively. The same parameters of the curd antigen were 90.9%（40/44）, 88.2%（15/17）, 0.79, and 60% （6/10） respectively. The sensitivity of purified antigen was significantly higher than the curd antigen. Conclusion Crude antigen of sheep hydatid cyst fluid was purified by molecular sieve chro- matography, and a Mr 55 000 nonspecific immunoreactive protein was identified and removed. The purification method used in this study provides clues for optimizing preparation of cyst antigen and quality control in development of effective diagnostics for eehinoeoccosis.

关 键 词：棘球蚴病 囊液抗原 分子筛层析 纯化 

分 类 号：R532.320.4[医药卫生—内科学]