棉铃虫甾醇载体蛋白2抑制剂的高通量筛选

发布时间：2017-12-28 05:10

本文关键词：棉铃虫甾醇载体蛋白2抑制剂的高通量筛选　出处：《湖北工业大学》2017年硕士论文　论文类型：学位论文

【摘要】：农业害虫对杀虫剂抗性的持续增加,严重威胁着农业可持续发展和人类生存。寻找新靶标,研制开发高效低毒环境友好型的新型农药是害虫防治重要急迫的研究任务。胆固醇是昆虫细胞膜的结构组分和蜕皮激素合成前体,然而昆虫自身并不能合成胆固醇。为满足正常生长、发育和繁殖需要,昆虫必须从食物中获取或通过共生微生物提供胆固醇。因此胆固醇的吸收转运对于昆虫的生存至关重要。甾醇载体蛋白2(sterol carrier protein-2,SCP-2)属于非特异性脂质转运蛋白,在昆虫体内参与胆固醇的吸收与转运。实验表明,用蚊子SCP-2蛋白抑制剂处理双翅目、鳞翅目、鞘翅目农业害虫幼虫可明显影响其生长发育,同时引起高成虫死亡率和低卵孵化率。SCP-2蛋白可作为新型农药靶标位点。本研究借助计算机辅助药物设计筛选出棉铃虫SCP-2蛋白小分子抑制剂,并对棉铃虫幼虫进行了生物测定。主要研究结果如下:(1)转化重组质粒pET-22b/HaSCP-2,在E.coli BL21(DE3)中诱导表达棉铃虫甾醇载体蛋白(HaSCP-2),最佳表达条件为:以0.2 mmol/L IPTG诱导重组质粒表达HaSCP-2蛋白,于25℃下恒温震荡培养10小时。HaSCP-2蛋白与底物结合活性分析表明:HaSCP-2蛋白与1,8-ANS结合常数Kd为1.973μmol/L;HaSCP-2蛋白与胆固醇结合半数有效剂量EC50为29.03μmol/L。(2)通过虚拟筛选技术,从218,780个小分子化合物中筛选出45种潜在的HaSCP-2蛋白抑制剂。根据化学结构多样性分类,从45中小分子化合物中挑选出9种不同结构骨架的化合物进行生物测定实验,最终得到5种具有较强杀虫效果的HaSCP-2蛋白抑制剂(H1、H7、H14、H19和H29)。对HaSCP-2蛋白抑制剂与HaSCP-2蛋白结合模式分析发现,HaSCP-2蛋白抑制剂在与HaSCP-2蛋白结合时为水平构象,抑制剂与HaSCP-2蛋白主要通过疏水力相互作用,抑制剂分子两端的苯环结构可与HaSCP-2蛋白的Tyr51、Phe53和Phe110氨基酸残基形成π-π堆叠相互作用,与Leu127、Thr128和Gln131氨基酸残基形成弱氢键等有利于提高抑制剂与HaSCP-2蛋白结合的稳定性。(3)生物测定结果显示:抑制剂(H1、H7、H14、H19和H29)在第11天对棉铃虫幼虫致死率分别为54%、50%、48%、33%和17%;随着抑制剂处理浓度升高,5种抑制剂处理棉铃虫幼虫体重下降14%~46%,抑制剂对棉铃虫生长抑制效果H1H14H19H29H7;以抑制剂处理棉铃虫幼虫,幼虫化蛹时间推迟3~5天;成虫产卵率下降12.5%~88.3%。环境毒性预测表明五种抑制剂为轻微或低的急性口服毒性,且对有益昆虫低毒。
[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.
【学位授予单位】：湖北工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S433

【参考文献】