荧光探针分子TNP-AMP识别蓝藻FBPase抑制剂作用位点的研究

发布时间：2018-01-07 10:14

  本文关键词：荧光探针分子TNP-AMP识别蓝藻FBPase抑制剂作用位点的研究　出处：《华中师范大学》2015年硕士论文　论文类型：学位论文

  更多相关文章： 果糖-1 6-二磷酸酶 蓝藻FBPase 荧光探针分子 TNP-AMP 结合位点 抑制剂筛选

【摘要】：蓝藻水华在中国分布广,对水生态系统、人体健康以及旅游经济均会造成严重危害。寻找高专一性、高效率又环境友好的杀藻剂,已经成为治理蓝藻水华的重要需求。研发有针对性、高专一性的抑制剂,不仅需要选择适宜的作用靶标,还需研究抑制剂与靶标的结合模式。蓝藻果糖-1,6-二磷酸酶(后简写为FBPase)在蓝藻体内含量极少,却在光合作用的Calvin循环中起到了至关重要的作用,而且其序列和结构与动植物体内所含FBPase存在着较大差异,故可作为杀藻剂的潜在作用靶标。抑制剂与蓝藻FBPase有两种不同的结合位点：底物位点和变构位点。探究抑制剂与蓝藻FBPase的结合位点,是研究其与蓝藻FBPase结合模式的基础。本文将荧光探针分子TNP-AMP与蓝藻FBPase结合,按照一定组合模式加入FBP、AMP和抑制剂,通过观察荧光的猝灭现象,建立了用于初步判断抑制剂结合位点的方法。本文主要进行了以下几方面工作：1.以pET-28a(+)为表达载体、以大肠杆菌E.Coli BL21(DE3)为表达菌株,对蓝藻FBPase进行了异源表达,并利用亲和层析方法对所得目的蛋白进行纯化,最终通过SDS-PAGE电泳验证其纯度,其产量为18.6 mg/L(培养基)。2.探索不同条件对荧光探针分子TNP-AMP结合蓝藻FBPase体系的荧光强度的影响,最终建立了TNP-AMP结合蓝藻FBPase荧光测定体系,具体条件为温度T=30℃,酸碱性pH=8.0,荧光探针分子TNP-AMP终浓度为12.9 μM,蓝藻FBPase最适终浓度为15μM,金属离子Mn2+最适终浓度为400 μM。3.探究了金属离子分别对单独的荧光染料或者蓝藻FBPase的荧光强度的影响,最终认为荧光探针分子TNP-AMP结合蓝藻FBPase的体系中加入金属离子之所以会引起荧光强度的增强,是因为金属离子能使蓝藻FBPase的构象发生一定程度的转变,从而与荧光探针分子TNP-AMP更好的结合。4.在前述荧光测定体系中,通过加入已知结合位点为底物位点的FBP和已知结合位点为变构位点的AMP两种化合物,观测荧光的猝灭,得出结论：荧光探针分子TNP-AMP不仅结合于蓝藻FBPase的变构位点,也结合于其底物位点。5.探索有望用于初步判断蓝藻FBPase抑制剂的结合位点的方法,并利用已知结合位点为底物位点的F6P化合物对此方法的准确性和可行性进行了验证。6.依次对hs, hx和x系列化合物以蓝藻FBPase为靶标进行初筛,通过分析初筛结果,选取x系列化合物中100μM终浓度下抑制率较高或结构比较有代表性的8个化合物进行抑制率的测定,其中x5抑制率最高,其IC50=2.3±0.2μM。通过分析实验结果,推测x系列化合物取代肼基上的一个三氟甲基被替换为乙氧基时,以及苯环上肼基取代的间位上取代基为吸电子基团时,有利于抑制率的提高。
[Abstract]:Cyanobacteria Shui Hua is widely distributed in China, which will cause serious harm to aquatic ecosystem, human health and tourism economy. Has become an important demand for the control of cyanobacteria Shui Hua. Research and development of targeted, one-sex inhibitors, not only need to select the appropriate target, but also need to study the combination of inhibitor and target model, cyanobacteria fructose -1. 6-diphosphatase (hereafter referred to as FB Pasein) is very small in cyanobacteria, but it plays an important role in photosynthesis Calvin cycle. Moreover, its sequence and structure are different from the FBPase contained in animals and plants. The inhibitor has two different binding sites to cyanobacteria FBPase: substrate site and metamorphic site, and explore the binding site between inhibitor and cyanobacteria FBPase. In this paper, the fluorescent probe molecule TNP-AMP was combined with cyanobacteria FBPase and added FBP according to a certain combination pattern. AMP and inhibitors were observed by fluorescence quenching. A method was established to determine the binding sites of inhibitors. In this paper, the following work was done: 1. PET-28a () was used as the expression vector. E. Coli BL21DE3) was used to express the FBPase of cyanobacteria, and the target protein was purified by affinity chromatography. The purity was verified by SDS-PAGE electrophoresis. The yield was 18.6 mg / L (medium. 2.) to explore the effect of different conditions on the fluorescence intensity of the fluorescent probe molecule TNP-AMP combined with cyanobacteria FBPase system. Finally, the fluorescence determination system of TNP-AMP combined with cyanobacteria FBPase was established. The specific conditions were as follows: temperature 30 鈩，

本文编号：1392110