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  本文關(guān)鍵詞：棉鈴蟲甾醇載體蛋白2抑制劑的高通量篩選　出處：《湖北工業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 甾醇載體蛋白-2(SCP-2) 抑制劑 虛擬篩選 殺蟲劑

【摘要】：農(nóng)業(yè)害蟲對殺蟲劑抗性的持續(xù)增加,嚴(yán)重威脅著農(nóng)業(yè)可持續(xù)發(fā)展和人類生存。尋找新靶標(biāo),研制開發(fā)高效低毒環(huán)境友好型的新型農(nóng)藥是害蟲防治重要急迫的研究任務(wù)。膽固醇是昆蟲細(xì)胞膜的結(jié)構(gòu)組分和蛻皮激素合成前體,然而昆蟲自身并不能合成膽固醇。為滿足正常生長、發(fā)育和繁殖需要,昆蟲必須從食物中獲取或通過共生微生物提供膽固醇。因此膽固醇的吸收轉(zhuǎn)運對于昆蟲的生存至關(guān)重要。甾醇載體蛋白2(sterol carrier protein-2,SCP-2)屬于非特異性脂質(zhì)轉(zhuǎn)運蛋白,在昆蟲體內(nèi)參與膽固醇的吸收與轉(zhuǎn)運。實驗表明,用蚊子SCP-2蛋白抑制劑處理雙翅目、鱗翅目、鞘翅目農(nóng)業(yè)害蟲幼蟲可明顯影響其生長發(fā)育,同時引起高成蟲死亡率和低卵孵化率。SCP-2蛋白可作為新型農(nóng)藥靶標(biāo)位點。本研究借助計算機(jī)輔助藥物設(shè)計篩選出棉鈴蟲SCP-2蛋白小分子抑制劑,并對棉鈴蟲幼蟲進(jìn)行了生物測定。主要研究結(jié)果如下:(1)轉(zhuǎn)化重組質(zhì)粒pET-22b/HaSCP-2,在E.coli BL21(DE3)中誘導(dǎo)表達(dá)棉鈴蟲甾醇載體蛋白(HaSCP-2),最佳表達(dá)條件為:以0.2 mmol/L IPTG誘導(dǎo)重組質(zhì)粒表達(dá)HaSCP-2蛋白,于25℃下恒溫震蕩培養(yǎng)10小時。HaSCP-2蛋白與底物結(jié)合活性分析表明:HaSCP-2蛋白與1,8-ANS結(jié)合常數(shù)Kd為1.973μmol/L;HaSCP-2蛋白與膽固醇結(jié)合半數(shù)有效劑量EC50為29.03μmol/L。(2)通過虛擬篩選技術(shù),從218,780個小分子化合物中篩選出45種潛在的HaSCP-2蛋白抑制劑。根據(jù)化學(xué)結(jié)構(gòu)多樣性分類,從45中小分子化合物中挑選出9種不同結(jié)構(gòu)骨架的化合物進(jìn)行生物測定實驗,最終得到5種具有較強(qiáng)殺蟲效果的HaSCP-2蛋白抑制劑(H1、H7、H14、H19和H29)。對HaSCP-2蛋白抑制劑與HaSCP-2蛋白結(jié)合模式分析發(fā)現(xiàn),HaSCP-2蛋白抑制劑在與HaSCP-2蛋白結(jié)合時為水平構(gòu)象,抑制劑與HaSCP-2蛋白主要通過疏水力相互作用,抑制劑分子兩端的苯環(huán)結(jié)構(gòu)可與HaSCP-2蛋白的Tyr51、Phe53和Phe110氨基酸殘基形成π-π堆疊相互作用,與Leu127、Thr128和Gln131氨基酸殘基形成弱氫鍵等有利于提高抑制劑與HaSCP-2蛋白結(jié)合的穩(wěn)定性。(3)生物測定結(jié)果顯示:抑制劑(H1、H7、H14、H19和H29)在第11天對棉鈴蟲幼蟲致死率分別為54%、50%、48%、33%和17%;隨著抑制劑處理濃度升高,5種抑制劑處理棉鈴蟲幼蟲體重下降14%~46%,抑制劑對棉鈴蟲生長抑制效果H1H14H19H29H7;以抑制劑處理棉鈴蟲幼蟲,幼蟲化蛹時間推遲3~5天;成蟲產(chǎn)卵率下降12.5%~88.3%。環(huán)境毒性預(yù)測表明五種抑制劑為輕微或低的急性口服毒性,且對有益昆蟲低毒。
[Abstract]:The continuous increase of insecticide resistance to agricultural pests is a serious threat to the sustainable development of agriculture and the survival of human beings. It is an important and urgent task for the pest control to find new targets and develop new pesticides with high efficiency and low toxicity and friendly environment. Cholesterol is a group of insect cell membrane structure and ecdysone biosynthesis precursor, but the insect itself does not synthesize cholesterol. In order to meet the needs of normal growth, development and reproduction, insects have to obtain or provide cholesterol from the food or through the symbiotic microorganism. Therefore, the absorption and transport of cholesterol is crucial to the survival of insects. Sterol carrier protein 2 (sterol carrier protein-2, SCP-2) is a non specific lipid transporter, which is involved in the absorption and transport of cholesterol in the insect. The experiment showed that mosquito SCP-2 protein inhibitor could significantly affect the growth and development of Diptera, Lepidoptera, Coleoptera agricultural pests, and cause high adult mortality and low egg hatching rate. SCP-2 protein can be used as a new target site for pesticide. With the help of computer aided drug design and screening of small molecule inhibitors of SCP-2 proteins of cotton bollworm, and the larvae of bioassay. The main results are as follows: (1) transformation of recombinant plasmid pET-22b/HaSCP-2 in E.coli BL21 (DE3) in cotton bollworm induced the expression of sterol carrier protein (HaSCP-2), the optimal expression conditions: expression of HaSCP-2 protein in 0.2 mmol/L IPTG induced by recombinant plasmid, at 25 C for Cuncussion cultured for 10 hours. The binding activity analysis of HaSCP-2 protein and substrate showed that the binding constant Kd of HaSCP-2 protein to 1,8-ANS was 1.973 mol/L, and the half effective dose of HaSCP-2 protein to cholesterol was 29.03 mol/L. (2) through the virtual screening technique, 45 potential HaSCP-2 protein inhibitors were screened from 218780 small molecular compounds. According to the classification of chemical structure diversity, 9 compounds with different structural skeletons were selected from 45 small and medium molecular compounds for bioassay. Finally, 5 kinds of HaSCP-2 protein inhibitors (H1, H7, H14, H19 and H29) with strong insecticidal efficacy were obtained. Model analysis found that the HaSCP-2 protein inhibitor combined with HaSCP-2 protein, HaSCP-2 protein inhibitor conformation is in combination with the HaSCP-2 protein level and HaSCP-2 protein inhibitor, mainly through hydrophobic interaction, benzene ring structure at both ends of the inhibitor molecules with HaSCP-2 protein Tyr51, Phe53 and Phe110 amino acid residues forming Pi Pi stacking interactions, and Leu127, Thr128 and Gln131 amino acid residues forming weak hydrogen bonds is conducive to enhance the stability of inhibitor binding to HaSCP-2 protein. (3) bioassay results showed that inhibitors (H1, H7, H14, H19 and H29) on the eleventh day of the cotton bollworm larvae mortality rate were 54%, 50%, 48%, 33% and 17%; with the inhibitor concentration, 5 inhibitors of cotton bollworm larvae decreased body weight 14%~46%, inhibitor of growth inhibitory effect of H1H14H19H29H7 on cotton bollworm; cotton bollworm larvae with inhibitors, pupation delayed 3~5 days; oviposition rate decreased by 12.5%~88.3%. Environmental toxicity prediction showed that the five inhibitors were mild or low in acute oral toxicity and low toxicity to beneficial insects.
【學(xué)位授予單位】：湖北工業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S433

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本文編號：1344682