食品与发酵工业 >

2019 , Vol. 45 >Issue 19: 17 - 24

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.020989

研究报告

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

  • 赵文平 ,
  • 贾龙刚 ,
  • 路福平 ,
  • 刘夫锋
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  • 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,天津 300457

修回日期: 2019-07-12

  网络出版日期: 2019-11-15

基金资助

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(18JCZDJC 33000)

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

  • ZHAO Wenping ,
  • JIA Longgang ,
  • LU Fuping ,
  • LIU Fufeng
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  • 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 University of Science & Technology, Tianjin 300457, China

Revised date: 2019-07-12

  Online published: 2019-11-15

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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。

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

本文引用格式

赵文平 , 贾龙刚 , 路福平 , 刘夫锋 . 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[J]. 食品与发酵工业, 2019 , 45(19) : 17 -24 . DOI: 10.13995/j.cnki.11-1802/ts.020989

Abstract

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.

Key words： Alzheimer’s disease (AD);; amyloid β protein 42 (Aβ42);; TEM1-β-lactamase (βlac);; expression of fusion protein; aggregation inhibitors; in vivo screening system of Escherichia coli

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