β-内酰胺酶表达及其基于荧光探针的抑制剂筛选体系研究

发布时间：2018-05-25 04:26

本文选题：β-内酰胺抗生素 + 细菌耐药性　；参考：《华东理工大学》2017年硕士论文

【摘要】：β-内酰胺类抗生素是目前治疗感染类疾病最为重要的药物之一,为人类的健康生活提供了重要的医疗保障。但是,随着这类抗生素的大量应用,临床上出现了越来越多的耐药菌,导致抗生素的药效大大降低,甚至出现了几乎能够抵抗所有抗生素的"超级细菌"。病菌产生耐药性的一个主要原因是细菌产生了一种能够水解破坏β-内酿胺类抗生素的酶,即β-内酰胺酶。现有研究表明,β-内酰胺类抗生素与相应的β-内酰胺酶抑制剂联合使用能够很大程度地恢复抗生素的抑菌效力。然而,对于金属主导的一类β-内酰胺酶目前还没有能够在临床中应用的β-内酰胺酶抑制剂,因此对于β-内酰胺酶抑制剂的研究仍然是当前非常重要的一个任务。目前β-内酰胺酶抑制剂的筛选主要是利用底物水解前后紫外吸收的差异,该方法所需底物量较大,紫外吸收波长短,易受到外界的干扰。在本论文中,我们通过对β-内酰胺酶基因的载体进行筛选和蛋白表达条件进行优化,最终完成了 8个不同类型的β-内酰胺酶的表达纯化,并应用部分β-内酰胺酶对荧光探针的水解进行了系统研究,通过相关条件优化完成了 β-内酰胺酶抑制剂快速筛选平台的构建。本论文建立基于β-内酰胺酶荧光探针的β-内酰胺酶抑制剂快速筛选平台,该平台利用荧光探针水解前后荧光强度的变化检测酶的水解活性,具有检测灵敏度高,酶的使用量少等优点,尤其适用于对潜在活性化合物的高通量筛选。
[Abstract]:尾-lactam antibiotics are currently one of the most important drugs for the treatment of infectious diseases, which provide an important medical guarantee for the healthy life of human beings. However, with the extensive application of these antibiotics, more and more drug-resistant bacteria appear in clinic, which leads to the decrease of antibiotic efficacy and even the emergence of "super bacteria" which can resist almost all antibiotics. One of the main reasons for bacterial resistance is that bacteria produce an enzyme that can hydrolyze and destroy 尾-lactamases, or 尾-lactamases. Current studies have shown that the combination of 尾-lactam antibiotics and corresponding 尾-lactamases inhibitors can restore the antimicrobial efficacy of antibiotics to a large extent. However, there is no 尾 -lactamase inhibitor that can be used in clinic, so the research of 尾 -lactamase inhibitor is still a very important task. At present, the screening of 尾 -lactamase inhibitors is mainly based on the difference of UV absorption before and after substrate hydrolysis. In this thesis, we selected the vector of 尾 -lactamase gene and optimized the expression conditions of 尾 -lactamases, and finally completed the purification of eight different types of 尾 -lactamases. Some 尾 -lactamases were used to study the hydrolysis of fluorescent probes, and the rapid screening platform of 尾 -lactamase inhibitors was constructed by optimizing the relevant conditions. In this paper, a rapid screening platform for 尾 -lactamase inhibitors based on fluorescence probe of 尾 -lactamase was established. The platform used fluorescence probe to detect the activity of 尾 -lactamase by using fluorescence probe before and after hydrolysis. It is especially suitable for high throughput screening of potential active compounds.
【学位授予单位】：华东理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R91

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