本文關(guān)鍵詞：熒光探針分子TNP-AMP識(shí)別藍(lán)藻FBPase抑制劑作用位點(diǎn)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(lán)藻FBPase 熒光探針分子 TNP-AMP 結(jié)合位點(diǎn) 抑制劑篩選

【摘要】：藍(lán)藻水華在中國(guó)分布廣,對(duì)水生態(tài)系統(tǒng)、人體健康以及旅游經(jīng)濟(jì)均會(huì)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(lán)藻水華的重要需求。研發(fā)有針對(duì)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(lán)藻果糖-1,6-二磷酸酶(后簡(jiǎn)寫為FBPase)在藍(lán)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(dòng)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(lán)藻FBPase有兩種不同的結(jié)合位點(diǎn)：底物位點(diǎn)和變構(gòu)位點(diǎn)。探究抑制劑與藍(lán)藻FBPase的結(jié)合位點(diǎn),是研究其與藍(lán)藻FBPase結(jié)合模式的基礎(chǔ)。本文將熒光探針分子TNP-AMP與藍(lán)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(diǎn)的方法。本文主要進(jìn)行了以下幾方面工作：1.以pET-28a(+)為表達(dá)載體、以大腸桿菌E.Coli BL21(DE3)為表達(dá)菌株,對(duì)藍(lán)藻FBPase進(jìn)行了異源表達(dá),并利用親和層析方法對(duì)所得目的蛋白進(jìn)行純化,最終通過SDS-PAGE電泳驗(yàn)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(duì)熒光探針分子TNP-AMP結(jié)合藍(lán)藻FBPase體系的熒光強(qiáng)度的影響,最終建立了TNP-AMP結(jié)合藍(lán)藻FBPase熒光測(cè)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(lán)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(duì)單獨(dú)的熒光染料或者藍(lán)藻FBPase的熒光強(qiáng)度的影響,最終認(rèn)為熒光探針分子TNP-AMP結(jié)合藍(lán)藻FBPase的體系中加入金屬離子之所以會(huì)引起熒光強(qiáng)度的增強(qiáng),是因?yàn)榻饘匐x子能使藍(lán)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(cè)定體系中,通過加入已知結(jié)合位點(diǎn)為底物位點(diǎn)的FBP和已知結(jié)合位點(diǎn)為變構(gòu)位點(diǎn)的AMP兩種化合物,觀測(cè)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(lán)藻FBPase的變構(gòu)位點(diǎn),也結(jié)合于其底物位點(diǎn)。5.探索有望用于初步判斷藍(lán)藻FBPase抑制劑的結(jié)合位點(diǎn)的方法,并利用已知結(jié)合位點(diǎn)為底物位點(diǎn)的F6P化合物對(duì)此方法的準(zhǔn)確性和可行性進(jìn)行了驗(yàn)證。6.依次對(duì)hs, hx和x系列化合物以藍(lán)藻FBPase為靶標(biāo)進(jìn)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(gè)化合物進(jìn)行抑制率的測(cè)定,其中x5抑制率最高,其IC50=2.3±0.2μM。通過分析實(shí)驗(yàn)結(jié)果,推測(cè)x系列化合物取代肼基上的一個(gè)三氟甲基被替換為乙氧基時(shí),以及苯環(huán)上肼基取代的間位上取代基為吸電子基團(tuán)時(shí),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(hào)：1392110