本文關(guān)鍵詞：熒光探針分子TNP-AMP識別藍藻FBPase抑制劑作用位點的研究　出處：《華中師范大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 果糖-1 6-二磷酸酶 藍藻FBPase 熒光探針分子 TNP-AMP 結(jié)合位點 抑制劑篩選

【摘要】：藍藻水華在中國分布廣,對水生態(tài)系統(tǒng)、人體健康以及旅游經(jīng)濟均會造成嚴(yán)重危害。尋找高專一性、高效率又環(huán)境友好的殺藻劑,已經(jīng)成為治理藍藻水華的重要需求。研發(fā)有針對性、高專一性的抑制劑,不僅需要選擇適宜的作用靶標(biāo),還需研究抑制劑與靶標(biāo)的結(jié)合模式。藍藻果糖-1,6-二磷酸酶(后簡寫為FBPase)在藍藻體內(nèi)含量極少,卻在光合作用的Calvin循環(huán)中起到了至關(guān)重要的作用,而且其序列和結(jié)構(gòu)與動植物體內(nèi)所含F(xiàn)BPase存在著較大差異,故可作為殺藻劑的潛在作用靶標(biāo)。抑制劑與藍藻FBPase有兩種不同的結(jié)合位點：底物位點和變構(gòu)位點。探究抑制劑與藍藻FBPase的結(jié)合位點,是研究其與藍藻FBPase結(jié)合模式的基礎(chǔ)。本文將熒光探針分子TNP-AMP與藍藻FBPase結(jié)合,按照一定組合模式加入FBP、AMP和抑制劑,通過觀察熒光的猝滅現(xiàn)象,建立了用于初步判斷抑制劑結(jié)合位點的方法。本文主要進行了以下幾方面工作：1.以pET-28a(+)為表達載體、以大腸桿菌E.Coli BL21(DE3)為表達菌株,對藍藻FBPase進行了異源表達,并利用親和層析方法對所得目的蛋白進行純化,最終通過SDS-PAGE電泳驗證其純度,其產(chǎn)量為18.6 mg/L(培養(yǎng)基)。2.探索不同條件對熒光探針分子TNP-AMP結(jié)合藍藻FBPase體系的熒光強度的影響,最終建立了TNP-AMP結(jié)合藍藻FBPase熒光測定體系,具體條件為溫度T=30℃,酸堿性pH=8.0,熒光探針分子TNP-AMP終濃度為12.9 μM,藍藻FBPase最適終濃度為15μM,金屬離子Mn2+最適終濃度為400 μM。3.探究了金屬離子分別對單獨的熒光染料或者藍藻FBPase的熒光強度的影響,最終認(rèn)為熒光探針分子TNP-AMP結(jié)合藍藻FBPase的體系中加入金屬離子之所以會引起熒光強度的增強,是因為金屬離子能使藍藻FBPase的構(gòu)象發(fā)生一定程度的轉(zhuǎn)變,從而與熒光探針分子TNP-AMP更好的結(jié)合。4.在前述熒光測定體系中,通過加入已知結(jié)合位點為底物位點的FBP和已知結(jié)合位點為變構(gòu)位點的AMP兩種化合物,觀測熒光的猝滅,得出結(jié)論：熒光探針分子TNP-AMP不僅結(jié)合于藍藻FBPase的變構(gòu)位點,也結(jié)合于其底物位點。5.探索有望用于初步判斷藍藻FBPase抑制劑的結(jié)合位點的方法,并利用已知結(jié)合位點為底物位點的F6P化合物對此方法的準(zhǔn)確性和可行性進行了驗證。6.依次對hs, hx和x系列化合物以藍藻FBPase為靶標(biāo)進行初篩,通過分析初篩結(jié)果,選取x系列化合物中100μM終濃度下抑制率較高或結(jié)構(gòu)比較有代表性的8個化合物進行抑制率的測定,其中x5抑制率最高,其IC50=2.3±0.2μM。通過分析實驗結(jié)果,推測x系列化合物取代肼基上的一個三氟甲基被替換為乙氧基時,以及苯環(huán)上肼基取代的間位上取代基為吸電子基團時,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，

本文編號：1392110