甜菜夜蛾拓扑异构酶Ⅰ氨基酸多型性对其活性的影响

发布时间：2018-06-02 09:43

本文选题：DNA拓扑异构酶Ⅰ + 定点突变　；参考：《中国农业科学院》2015年硕士论文

【摘要】：DNA拓扑异构酶I(Top Ⅰ)参与机体内的DNA复制、转录和重组反应,是喜树碱类化合物的作用靶标,其结构变化会影响对喜树碱类抑制剂的敏感性。本课题组通过对已知昆虫TopⅠ氨基酸进行比对后,发现昆虫TopⅠ在420,530,653和729四个位点存在多型性,这种多型性是否会影响昆虫对喜树碱类抑制剂的敏感性未见有报道。本研究通过对甜菜夜蛾TopⅠ基因进行定点突变、诱导表达及纯化,研究了氨基酸多型性对TopⅠ解旋活性以及对喜树碱类抑制剂敏感性的影响,并以TopⅠ为靶标进行了非喜树碱类抑制剂的初步筛选。取得如下研究结果：完全重叠PCR能实现甜菜夜蛾TopⅠ定点突变,无其他突变引入。在选择的浓度范围内,IPTG均能成功诱导目的蛋白表达,且表达量随着IPTG浓度的增加而增加,但在相同浓度IPTG诱导下,突变蛋白的表达量较野生型明显减少。氨基酸多型性对甜菜夜蛾TopⅠ与钾离子的亲和性及解旋活性有影响。相同条件下,与野生型蛋白相比,突变蛋白对钾离子的亲和性均有所增强,启动解旋反应时的钾离子浓度较野生型蛋白低,最适钾离子浓度也有所改变。在最适钾离子浓度下,对DNA的解旋活性也存在差异。其中,L530P突变蛋白的最适钾离子浓度为100mM,较野生型蛋白的最适钾离子浓度(150mmM)降低了30%,但两者在最适钾离子浓度下的催化活力一致,比活力均为128×109U/mg pro;A653T突变蛋白对钾离子的适宜浓度范围增宽,在100 mM到200 mM范围内的解旋活性均超过80%,但比活力较野生型蛋白降低了50%,为6.40×108 U/mg pro; S729T突变蛋白的最适钾离子浓度与野生型蛋白相同,为150 mM,但催化活力较野生蛋白下降了75%,比活力为3.20×108U/mg pro。氨基酸多型性改变了甜菜夜蛾TopⅠ对喜树碱类抑制剂的敏感性。同等条件下,在所选定的浓度范围内,L530P突变蛋白对伊立替康和拓扑替康完全丧失了敏感性,对喜树碱和羟基喜树碱的敏感性临界浓度均为7.5 μM,在此临界浓度下,对羟基喜树碱的敏感性与野生型蛋白相当,对喜树碱的敏感度下降了60%。A653T突变蛋白对喜树碱、羟基喜树碱、伊立替康和拓扑替康的敏感性临界浓度分别为5.0μM、7.5μM、7.5μM和10.0μM,在此临界浓度下,对伊立替康的敏感度较野生蛋白提高了28%,对喜树碱、羟基喜树碱和拓扑替康的敏感度则分别下降了40%、70%和80%；S729T突变蛋白对伊立替康和拓扑替康完全不敏感,对喜树碱和羟基喜树碱的敏感性临界浓度分别为0.5 μM和2.5州,在此临界浓度下,对喜树碱和羟基喜树碱的敏感度分别下降了17%和24%。氨基酸多型性对甜菜夜蛾TopⅠ与喜树碱和羟基喜树碱的结合速率亦有影响。在敏感性临界浓度下,酶与喜树碱和羟基喜树碱充分结合所用时间存在差异。与野生型蛋白相比,L530P突变蛋白与喜树碱的结合速率下降,酶的解旋活性依然存在,并在4 min时对体系内超螺旋DNA 100%解旋,而在野生型蛋白体系内,酶活性被100%抑制；但L530P突变蛋白与羟基喜树碱的结合速率增加,在所测定时间范围内,酶完全失去解旋活性。A653T和S729T突变蛋白与喜树碱的结合速率下降,完全消耗体系内喜树碱所用的时间分别为15 min和8min,是野生型蛋白(4min)的3.75倍和2倍；A653T与羟基喜树碱的结合速率增加,充分结合时间为2 min,较野生型蛋白(4min)提前了2min; S729T与羟基喜树碱充分结合所用时间与野生型蛋白一致,均为4 min,但突变蛋白的羟基喜树碱处理浓度(2.5μM)较野生型蛋白(5.0μM)低,因而,结合速率有所下降。通过DNA超螺旋解旋法,测定了羟基喜树碱氯乙基异氰酸酯取代物对突变前后蛋白解旋活性的影响,发现该化合物对野生型蛋白保留了与其母体化合物羟基喜树碱的抑制效果,对L530P和A653T突变蛋白的抑制率较母体化合物有所降低,最大抑制效率分别为20%和16%；对S729T的抑制效果则较母体化合物增加,最大抑制效率为100%。利用体外酶活性检测体系,测定了辛可宁,利血平,肉叶云香碱,育亨宾盐酸盐,1-(3,4-二甲氧基苄基)-6,7-二-氧基异喹啉盐酸盐,3-二甲基氨基甲基吲哚,阿托品,阿马里新,盐酸小檗碱,槲皮素和槐定碱11种化合物对拓扑异构酶解旋活性的影响,结果发现这些化合物对TopⅠ均无抑制活性。综上所述,当甜菜夜蛾TopⅠ氨基酸序列中特定位点被替换后,酶的解旋活性和对抑制剂敏感性均发生相应改变。深入开展氨基酸多型性与昆虫TopⅠ对抑制剂敏感性之间的效应关系研究,利用拓扑异构酶体外表达系统,和酶活性检测体系,开发出更多的以’Top Ⅰ为靶标的抑制剂先导化合物,推动新农药创制和应用。
[Abstract]:DNA topoisomerase I (Top I) participates in DNA replication, transcriptional and recombination reactions in the body, which is the target of the action of camptothecin compounds, and their structural changes affect the sensitivity to camptothecin inhibitors. By comparing the known insect Top I amino acids, the group found that the insect Top I existed at four and 729 sites at 420530653 and 729 sites. In this study, the effect of amino acid polymorphism on the activity of Top I and the sensitivity to camptothecin inhibitors was studied by the fixed-point mutation of the Top I gene of the Spodoptera beetle, and the effect of the amino acid polymorphism on the sensitivity of the camptothecin inhibitor. The target was Top I as a target. The preliminary screening of non camptothecin inhibitors was carried out. The results are as follows: complete overlap of PCR can achieve fixed point mutation of the beet armyworm, Top I, without other mutations. Within the range of selected concentrations, IPTG can successfully induce the expression of the target protein, and the expression increases with the increase of the concentration of IPTG, but is induced at the same concentration of IPTG. The expression of mutant protein was significantly lower than that in the wild type. Amino acid polymorphism had an effect on the affinity and spin activity of Top I and potassium ion in the beet armyworm. Under the same condition, the affinity of the mutant protein to potassium ion was enhanced, and the concentration of potassium ion was lower than that of wild type protein. The concentration of K + is also changed. Under the optimum potassium ion concentration, there are also differences in the spin activity of DNA. The optimum potassium concentration of L530P mutant protein is 100mM, and the optimum potassium ion concentration (150mmM) of the wild type protein is reduced by 30%, but the catalytic activity of both of them is consistent with the optimum potassium concentration, and the specific activity is 128 x 10. 9U/mg Pro; A653T mutation protein broadened the suitable concentration range for potassium ion, and the spin activity in the range of 100 mM to 200 mM was more than 80%, but the activity was 50% lower than that of wild type protein and 6.40 x 108 U/mg Pro; the optimum potassium concentration of S729T mutant protein was the same as that of wild type egg white, 150 mM, but the catalytic activity was more than that of wild protein. The sensitivity of Top I to camptothecin inhibitors was reduced by 75% and the activity of 3.20 x 108U/mg pro. amino acids. Under the same condition, the L530P mutant protein completely lost sensitivity to irinotecan and topotecan, and the critical concentration of the sensitivity to xerotin and hydroxycamptothecin was all At this critical concentration, the sensitivity of HCPT was equivalent to that of wild type protein at this critical concentration. The sensitivity to camptothecin was reduced by the sensitivity of 60%.A653T mutation to camptothecin, hydroxycamptothecin, irinotecan and topotecan, respectively, at the critical concentration of 5 u M, 7.5 M, 7.5 mu M and 10 micron M at this critical concentration, to erinecan at this critical concentration. The sensitivity of the wild protein was 28% higher than that of the wild protein, and the sensitivity to camptothecin, hydroxycamptothecin and topotecan decreased by 40%, 70% and 80%, respectively. The S729T mutant protein was completely insensitive to irinotecan and topotecan, and the critical concentration of camptothecin and hydroxycamptothecin was 0.5 mu M and 2.5 state respectively, at this critical concentration, The sensitivity of camptothecin and hydroxycamptothecin decreased by 17% and 24%. amino acid polymorphism respectively on the binding rate of Top I with camptothecin and hydroxycamptothecin. At the critical concentration, the time used to combine the enzyme with camptothecin and hydroxycamptothecin was different. The L530P mutant egg was compared with the wild type protein. The binding rate of white and camptothecin decreased, the activity of the enzyme still existed, and the super spiral DNA 100% in the system was rotated at 4 min, while in the wild type protein system, the enzyme activity was inhibited by 100%, but the binding rate of L530P mutant protein and hydroxycamptothecin increased, and the enzyme completely lost the activity of spin activity.A653T and S within the determined time range. The binding rate of 729T mutant protein and camptothecin decreased. The time used for camptothecin in full consumption system was 15 min and 8min, respectively, 3.75 times and 2 times that of wild type protein (4min). The binding rate of A653T and hydroxycamptothecin increased, the time was 2 min, and 4min was ahead of 2min; S729T and hydroxycamptothecin were filled. The time used to combine with the wild type protein was 4 min, but the concentration of Hydroxycamptothecin (2.5 M) of the mutant protein was lower than that of the wild type protein (5 mu M). Therefore, the binding rate decreased. The effect of hydroxycamptothecin chloro ethyl isocyanate substituents on the protein spin activity before and after the mutation was measured by DNA super spiral method. The inhibitory effect of the compound on the wild-type protein was found. The inhibition rate of L530P and A653T mutant protein was lower than that of the parent compound, and the maximum inhibitory efficiency was 20% and 16%, respectively. The inhibition effect on S729T was higher than that of the mother compound, and the maximum inhibition efficiency was 100%. using in vitro enzyme. The activity detection system was used to determine the effects of 11 kinds of compounds on topospin activity of topoisomerase, such as cindine, reserpine, meatropine, yohimbine hydrochloride, 1- (3,4- two methoxy benzyl) -6,7- two - isoquinoline hydrochloride, 3- two methylamino methyl indole, atropine, amayin, berberine, quercetin and Sophora japonicine. These compounds have no inhibitory activity on Top I. To sum up, when the special location of the Top I amino acid sequence of the beet armyworm Spodoptera Spodoptera is replaced, the activity of the enzyme and the sensitivity of the inhibitor all change accordingly. The relationship between the effect of the amino acid polymorphism and the sensitivity of the insect Top I to the inhibitor is studied, and the topoisomerase is used. In vitro expression system and enzyme activity detection system have developed more lead compounds with "Top I" as target inhibitors to promote the creation and application of new pesticides.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433.4

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