基于β-内酰胺酶构建大肠杆菌体内Aβ42聚集抑制剂筛选体系  

Construction of an in vivo system of Escherichia coli for screening Aβ42 aggregation inhibitors based on β-lactamase

作　　者：赵文平 贾龙刚 路福平[1] 刘夫锋 ZHAO Wenping;JIA Longgang;LU Fuping;LIU Fufeng(Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, National Engineering Laboratory for Industrial Enzymes,Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin Universityof Science & Technology, Tianjin 300457, China)

机构地区：[1]工业发酵微生物教育部重点实验室天津市工业微生物重点实验室工业酶国家工程实验室天津科技大学生物工程学院

出　　处：《食品与发酵工业》2019年第19期17-24,共8页Food and Fermentation Industries

基　　金：国家自然科学基金面上项目(21878234、21576199);天津市应用基础及前沿技术研究计划重点项目(18JCZDJC33000)

摘　　要：淀粉样β蛋白(Aβ)的错误折叠和聚集是导致阿尔茨海默病(Alzheimer’s disease, AD)的主要原因,开发Aβ聚集抑制剂对于缓解和治疗AD至关重要。根据TEM1-β-内酰胺酶(TEM1-β-lactamase,βlac)的结构特征及Aβ42的聚集特性,利用分子生物学方法将Aβ42插入到βlac 196-197氨基酸残基之间,构建了BL21-βlac-Aβ42重组菌株。利用已知Aβ42聚集抑制剂,分别通过斑点滴定实验、平板涂布实验和菌体浓度变化检测实验进行了验证。结果显示,添加已知Aβ42抑制剂后,BL21-βlac-Aβ42菌株在特定氨苄青霉素(ampicillin, Amp)浓度条件下生长状况得到改善,允许微生物细胞生长的最大细胞稀释倍数增大,证明该融合体系能应用于大肠杆菌体内筛选Aβ42聚集抑制剂。该研究实现了淀粉样蛋白聚集抑制剂在微生物体内的高通量筛选,为开发新型抗Aβ聚集药物奠定了基础。Misfolding and aggregation of amyloid β protein (Aβ) play a key role in developing Alzheimer's disease (AD). Therefore, establishing aggregation inhibitors of Aβ is important for alleviating and treating AD. Based on the structural property of TEM1-β-lactamase (βlac) and the aggregation characteristics of Aβ42, this study inserted Aβ42 between residue 196 and 197 of βlac to construct BL21-βlac-Aβ42 recombinant strain. Moreover, it was identified using several known Aβ42 inhibitors. The results showed that with the addition of known Aβ42 inhibitors, the growth of recombinant BL21-βlac-Aβ42 was improved in ampicillin with specified concentration, and the maximum cell dilution for cell growth was also increased. Overall, this system realized high-throughput screening of amyloid aggregation inhibitors in microorganisms, which is helpful for developing new anti-Aβ aggregation drugs.

关 键 词：阿尔茨海默病 淀粉样β蛋白42 (Aβ42) TEM1-β-内酰胺酶(βlac) 融合蛋白表达 聚集抑制剂 大肠杆菌体内筛选系统 

分 类 号：R91[医药卫生—药学]