β分泌酶抑制剂体外筛选模型的构建与验证  

A screen assay of β-secretase inhibitors in vitro

作　　者：赵彬[1] 杨红军[2] 赵晶[1] 

机构地区：[1]成都军区昆明总医院医学实验科,云南昆明650032 [2]昆明医学院第一附属医院,云南昆明650031

出　　处：《中国药理学通报》2006年第1期121-123,共3页Chinese Pharmacological Bulletin

基　　金：云南省自然科学基金资助项目(No2002C0023Q)

摘　　要：目的构建β分泌酶抑制剂筛选模型用于治疗阿尔采末病药物筛选。方法克隆人β分泌酶基因,并在大肠杆菌中表达,经纯化、复性后获得重组蛋白。以重组β淀粉样肽融合蛋白为底物,体外建立酶学反应。酶切反应液用聚丙烯酰胺凝胶电泳检测,或将其包被金属螯和酶标板,用抗β淀粉样肽抗体行ELISA检测。结果用RT-PCR法克隆了人β分泌酶基因,构建入表达载体,诱导表达后获得高效表达。建立体外酶学反应,用ELISA和SDS-PAGE检测,二者检测结果一致,且用所构建方法测得阳性抑制剂的IC50值与文献报道吻合。对77种用于老年痴呆治疗的中草药提取物进行筛选,仅发现有17种显示微弱的β分泌酶抑制活性。结论成功构建了β分泌酶抑制剂筛选模型并用于中草药提取物活性筛选。Aim To construct a screen assay of β-secretase inhibitors. Methods A human β-seeretase eDNA was cloned by RT-PCR and then reconstructed in an expression vector. The recombinant β-seeretase fusion protein was expressed and purified from Eseheriehia coli. A screen assay of β-secretase inhibitors was constructed in vitro using a recombinant A β-fusion protein as substrate. The enzyme reaction was detected by SDS-PAGE and an ELISA method using metal chelating plates to catch the substrate. Results A human β-seeretaae eDNA was cloned and expressed in escheriehia coll. The β-secretase enzyme reaction can be detected by two kinds of method： SDS-PAGE and ELISA. The IC50 for an inhibitor statVal determined by this method was in good agreement with those reported in the literature. Seventy seven species of Chinese traditional medical plants were screened using this method and seventeen species show weak inhibitory activity. Conclusion A screen assay of β-secretase inhibitors was successfully constructed and can be used to screen natural products.

关 键 词：Β分泌酶 筛选模型 阿尔采末病 ELISA 

分 类 号：R341[医药卫生—基础医学] R394.2