本文選題：DNA拓?fù)洚悩?gòu)酶Ⅰ + 定點(diǎn)突變�。� 參考：《中國(guó)農(nóng)業(yè)科學(xué)院》2015年碩士論文

【摘要】：DNA拓?fù)洚悩?gòu)酶I(Top Ⅰ)參與機(jī)體內(nèi)的DNA復(fù)制、轉(zhuǎn)錄和重組反應(yīng),是喜樹堿類化合物的作用靶標(biāo),其結(jié)構(gòu)變化會(huì)影響對(duì)喜樹堿類抑制劑的敏感性。本課題組通過對(duì)已知昆蟲TopⅠ氨基酸進(jìn)行比對(duì)后,發(fā)現(xiàn)昆蟲TopⅠ在420,530,653和729四個(gè)位點(diǎn)存在多型性,這種多型性是否會(huì)影響昆蟲對(duì)喜樹堿類抑制劑的敏感性未見有報(bào)道。本研究通過對(duì)甜菜夜蛾TopⅠ基因進(jìn)行定點(diǎn)突變、誘導(dǎo)表達(dá)及純化,研究了氨基酸多型性對(duì)TopⅠ解旋活性以及對(duì)喜樹堿類抑制劑敏感性的影響,并以TopⅠ為靶標(biāo)進(jìn)行了非喜樹堿類抑制劑的初步篩選。取得如下研究結(jié)果：完全重疊PCR能實(shí)現(xiàn)甜菜夜蛾TopⅠ定點(diǎn)突變,無其他突變引入。在選擇的濃度范圍內(nèi),IPTG均能成功誘導(dǎo)目的蛋白表達(dá),且表達(dá)量隨著IPTG濃度的增加而增加,但在相同濃度IPTG誘導(dǎo)下,突變蛋白的表達(dá)量較野生型明顯減少。氨基酸多型性對(duì)甜菜夜蛾TopⅠ與鉀離子的親和性及解旋活性有影響。相同條件下,與野生型蛋白相比,突變蛋白對(duì)鉀離子的親和性均有所增強(qiáng),啟動(dòng)解旋反應(yīng)時(shí)的鉀離子濃度較野生型蛋白低,最適鉀離子濃度也有所改變。在最適鉀離子濃度下,對(duì)DNA的解旋活性也存在差異。其中,L530P突變蛋白的最適鉀離子濃度為100mM,較野生型蛋白的最適鉀離子濃度(150mmM)降低了30%,但兩者在最適鉀離子濃度下的催化活力一致,比活力均為128×109U/mg pro;A653T突變蛋白對(duì)鉀離子的適宜濃度范圍增寬,在100 mM到200 mM范圍內(nèi)的解旋活性均超過80%,但比活力較野生型蛋白降低了50%,為6.40×108 U/mg pro; S729T突變蛋白的最適鉀離子濃度與野生型蛋白相同,為150 mM,但催化活力較野生蛋白下降了75%,比活力為3.20×108U/mg pro。氨基酸多型性改變了甜菜夜蛾TopⅠ對(duì)喜樹堿類抑制劑的敏感性。同等條件下,在所選定的濃度范圍內(nèi),L530P突變蛋白對(duì)伊立替康和拓?fù)涮婵低耆珕适Я嗣舾行?對(duì)喜樹堿和羥基喜樹堿的敏感性臨界濃度均為7.5 μM,在此臨界濃度下,對(duì)羥基喜樹堿的敏感性與野生型蛋白相當(dāng),對(duì)喜樹堿的敏感度下降了60%。A653T突變蛋白對(duì)喜樹堿、羥基喜樹堿、伊立替康和拓?fù)涮婵档拿舾行耘R界濃度分別為5.0μM、7.5μM、7.5μM和10.0μM,在此臨界濃度下,對(duì)伊立替康的敏感度較野生蛋白提高了28%,對(duì)喜樹堿、羥基喜樹堿和拓?fù)涮婵档拿舾卸葎t分別下降了40%、70%和80%；S729T突變蛋白對(duì)伊立替康和拓?fù)涮婵低耆幻舾?對(duì)喜樹堿和羥基喜樹堿的敏感性臨界濃度分別為0.5 μM和2.5州,在此臨界濃度下,對(duì)喜樹堿和羥基喜樹堿的敏感度分別下降了17%和24%。氨基酸多型性對(duì)甜菜夜蛾TopⅠ與喜樹堿和羥基喜樹堿的結(jié)合速率亦有影響。在敏感性臨界濃度下,酶與喜樹堿和羥基喜樹堿充分結(jié)合所用時(shí)間存在差異。與野生型蛋白相比,L530P突變蛋白與喜樹堿的結(jié)合速率下降,酶的解旋活性依然存在,并在4 min時(shí)對(duì)體系內(nèi)超螺旋DNA 100%解旋,而在野生型蛋白體系內(nèi),酶活性被100%抑制；但L530P突變蛋白與羥基喜樹堿的結(jié)合速率增加,在所測(cè)定時(shí)間范圍內(nèi),酶完全失去解旋活性。A653T和S729T突變蛋白與喜樹堿的結(jié)合速率下降,完全消耗體系內(nèi)喜樹堿所用的時(shí)間分別為15 min和8min,是野生型蛋白(4min)的3.75倍和2倍；A653T與羥基喜樹堿的結(jié)合速率增加,充分結(jié)合時(shí)間為2 min,較野生型蛋白(4min)提前了2min; S729T與羥基喜樹堿充分結(jié)合所用時(shí)間與野生型蛋白一致,均為4 min,但突變蛋白的羥基喜樹堿處理濃度(2.5μM)較野生型蛋白(5.0μM)低,因而,結(jié)合速率有所下降。通過DNA超螺旋解旋法,測(cè)定了羥基喜樹堿氯乙基異氰酸酯取代物對(duì)突變前后蛋白解旋活性的影響,發(fā)現(xiàn)該化合物對(duì)野生型蛋白保留了與其母體化合物羥基喜樹堿的抑制效果,對(duì)L530P和A653T突變蛋白的抑制率較母體化合物有所降低,最大抑制效率分別為20%和16%；對(duì)S729T的抑制效果則較母體化合物增加,最大抑制效率為100%。利用體外酶活性檢測(cè)體系,測(cè)定了辛可寧,利血平,肉葉云香堿,育亨賓鹽酸鹽,1-(3,4-二甲氧基芐基)-6,7-二-氧基異喹啉鹽酸鹽,3-二甲基氨基甲基吲哚,阿托品,阿馬里新,鹽酸小檗堿,槲皮素和槐定堿11種化合物對(duì)拓?fù)洚悩?gòu)酶解旋活性的影響,結(jié)果發(fā)現(xiàn)這些化合物對(duì)TopⅠ均無抑制活性。綜上所述,當(dāng)甜菜夜蛾TopⅠ氨基酸序列中特定位點(diǎn)被替換后,酶的解旋活性和對(duì)抑制劑敏感性均發(fā)生相應(yīng)改變。深入開展氨基酸多型性與昆蟲TopⅠ對(duì)抑制劑敏感性之間的效應(yīng)關(guān)系研究,利用拓?fù)洚悩?gòu)酶體外表達(dá)系統(tǒng),和酶活性檢測(cè)體系,開發(fā)出更多的以’Top Ⅰ為靶標(biāo)的抑制劑先導(dǎo)化合物,推動(dòng)新農(nóng)藥創(chuàng)制和應(yīng)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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S433.4

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本文編號(hào)：1968306