幽门螺杆菌尿素酶的提取与纯化  

Extraction and Purification of Urease from Helicobacter pylori

作　　者：聂作明[1] 杨严俊[1] 徐榕榕[1] 盛剑俊[1] 

机构地区：[1]江南大学食品学院生物技术组

出　　处：《食品与发酵工业》2004年第5期40-45,共6页Food and Fermentation Industries

摘　　要：采用2种方法分离纯化了幽门螺杆菌尿素酶。第1种方法是以 DEAE-sepharose FF离子交换剂和Superdex 200凝胶为分离介质,用 NaCl盐浓度梯度洗脱法从幽门螺杆菌超声上清液中提取纯化尿素酶抗原,所得到的纯化尿素酶经SDS-PAGE电泳分析,表明它具有很高的纯度,只含有分子质量为64 000 u和31 000 u两种蛋白成分,分别对应于幽门螺杆菌尿素酶的A、B两种亚基。第2种方法是以 DEAE-sepharose FF离子交换剂为分离介质,采用先去掉部分杂蛋白的方法纯化尿素酶,其中的去杂步骤操作简单、快捷且处理样品量大,大大减轻了后面纯化步骤的难度,适合于尿素酶的大量制备。纯化的尿素酶作为抗原采用ELISA间接法检测抗HP蛋黄免疫球蛋白(IgY),实验表明,其仍具有良好的抗原性,是HP主要抗原物质。应用纯化尿素酶抗原或纯化尿素酶抗原与超声粉碎抗原的混合抗原刺激蛋鸡应该能够产生抗HP效价更高的蛋黄免疫球蛋白,可以将其应用于保健或各种与HP相关的胃病的抗菌免疫治疗。The Helicobacter pylori (HP) urease was purified by two methods in this research. One is that the enzyme was purified by DEAE - Sepharose Fast Flow with a linear NaCl-gradient and Su- perdex 200 chromatographies. There are two bands shown at 65ku and 31 ku on SDS - PAGE. In the second way, we removed the major contaminants in supernatant prior to the ion exchnge separation to facilitate the following purification steps; and the enzyme was purified by DEAE - Separose Fast Flow. This purified urease had been used as the ELISA coating antigen to detect the specific activity of anti - HP immunoglobulin prepared from the egg yolk(IgY), the results showed that it has good anti- genicity. The anti - HP IgY with active activities can be obtained after immunization with HP urease or mixture of HP and urease antigen. It can be applied to human healthcare purpose and it can be also used for antibacterial immune treatment for gastric disease associated with HP.

关 键 词：幽门螺杆菌 尿素酶 提取 纯化 NaCI盐浓度梯度洗脱法 检测 抗HP蛋黄免疫球蛋白 抗原 

分 类 号：TQ464[化学工程—制药化工]