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  本文關(guān)鍵詞： 定向固定化 β_2-腎上腺素受體 相互作用 活性篩選 蛋白質(zhì)構(gòu)象　出處：《西北大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：功能蛋白質(zhì)與小分子配體的相互作用研究對于揭示生命活動過程所涉及的復(fù)雜信號傳遞和開發(fā)高效創(chuàng)新藥物具有重要意義。以固定化功能蛋白質(zhì)為核心的生物傳感器、試劑盒、酶反應(yīng)器和親和色譜方法是實現(xiàn)上述研究的主要途徑。然而,由于固定化界面蛋白質(zhì)取向和構(gòu)象雜亂不一,使得其在功能蛋白質(zhì)與藥物相互作用研究等方面的測定結(jié)果偏離生理狀態(tài)。因此,固定化功能蛋白質(zhì)的取向和構(gòu)象調(diào)控方法研究就成為醫(yī)藥、化學(xué)和生物學(xué)領(lǐng)域的前沿研究問題之一。作者所在實驗室在前期的工作中,建立了β2-腎上腺素受體色譜方法,證明方法可用于受體與藥物的相互作用研究和藥物活性成分篩選。基于此,本論文針對色譜固定相表面受體取向和構(gòu)象雜亂不一的問題,圍繞固定化β2-腎上腺素受體取向和構(gòu)象誘導(dǎo)、調(diào)控方法的建立及應(yīng)用展開系統(tǒng)研究。該研究有望為其他以固定化功能蛋白質(zhì)為核心的高靈敏方法的建立提供依據(jù),對高效創(chuàng)新藥物的開發(fā)具有重要意義。全文共分4章,作者的主要貢獻(xiàn)如下：(1)建立了一種可逆的β2-腎上腺素受體定向固定化新方法,構(gòu)建了β2-腎上腺素受體取向及構(gòu)象調(diào)控模型。利用鎳離子與β2-腎上腺素受體結(jié)構(gòu)中組氨酸標(biāo)簽所含高密度咪唑基之間的特異性作用,將該受體可逆的定向固定在大孔硅膠表面,制備了定向固定化的β2-腎上腺素受體色譜固定相。結(jié)果表明,該方法可獲得取向統(tǒng)一的固定化受體。以工具藥鹽酸氯丙那林等為配體,對受體色譜模型的特異性和穩(wěn)定性進(jìn)行了表征。在此基礎(chǔ)上,以鹽酸麻黃堿和鹽酸偽麻黃堿為構(gòu)象依賴探針,通過改變色譜柱溫度、流動相組成以及配體條件等對固定化受體的構(gòu)象進(jìn)行調(diào)控。結(jié)果表明：當(dāng)色譜柱溫度為25.0℃時,可獲得固定化受體的最穩(wěn)定構(gòu)象態(tài)：當(dāng)流動相磷酸鹽緩沖溶液濃度為5.0mmol/L(pH=7.40)時,固定化β2-腎上腺素受體構(gòu)象最優(yōu)；以(S)-普萘洛爾為配體,考察了藥物配體對固定化β2-腎上腺素受體構(gòu)象的影響,證明藥物配體可誘導(dǎo)固定化受體構(gòu)象發(fā)生變化,且濃度為5.0nmol/L的(S)-普萘洛爾可使固定化β2-腎上腺素受體獲得最優(yōu)構(gòu)象。這方面的研究為其他固定化功能蛋白質(zhì)取向及構(gòu)象的調(diào)控提供了方法學(xué)借鑒。(2)應(yīng)用穩(wěn)定構(gòu)象態(tài)β2-腎上腺素受體模型研究了受體與硫酸特布他林等七種藥物配體的相互作用,為明確藥物的作用機(jī)制提供了依據(jù)。采用前沿色譜法測定了硫酸特布他林、鹽酸甲氧那明、鹽酸氯丙那林、鹽酸妥布特羅、硫酸沙丁胺醇、鹽酸克倫特羅和鹽酸班布特羅與β2-腎上腺素受體相互作用的結(jié)合常數(shù)和結(jié)合位點數(shù)。結(jié)果表明：七種藥物和β2-腎上腺素受體均存在一類結(jié)合位點,結(jié)合常數(shù)依次為：1.84x104 M-1,1.71×104 M-1,2.3×1O4M-1,2.09×104M-1,1.98×104 M-1,2.37×104M-1和9.43×104 M-1；結(jié)合位點數(shù)依次為：8.3×10-4M,3.88×10-5M, 1.02×10-5M,2.16×10-5M,7.5×10-5M,1.82×10-5M, 2.47×10-5M。采用微透析法對上述結(jié)果進(jìn)行了驗證,表明兩種方法所得結(jié)合常數(shù)和結(jié)合位點數(shù)在數(shù)量級上相同,證明β2-腎上腺素受體色譜模型能用于研究藥物分子與受體的相互作用,為在線蛋白質(zhì)-藥物相互作用高通量分析提供了方法學(xué)參考。(3)應(yīng)用穩(wěn)定構(gòu)象態(tài)β2-腎上腺素受體色譜模型對延胡索和百部中的活性成分進(jìn)行了篩選,采用前沿色譜和競爭置換色譜法對活性成分與受體的作用機(jī)制進(jìn)行了探討。加熱回流法制備延胡索和百部的總提物,受體色譜進(jìn)行分析,收集保留成分經(jīng)反相液相色譜-質(zhì)譜聯(lián)用法進(jìn)行結(jié)構(gòu)鑒定。結(jié)果發(fā)現(xiàn),四氫小檗堿、原阿片堿、紫堇堿和四氫非洲防己堿為延胡索中與β2-腎上腺素受體有特異性作用的活性成分；百部新堿為百部中作用于β2-腎上腺素受體的活性成分。進(jìn)一步采用前沿色譜法測得四氫小檗堿和原阿片堿與β2-AR的結(jié)合常數(shù)分別為9.04×104M-1和4.30×104 M-1,結(jié)合位點數(shù)分別為6.67×10-4M和5.88×10-4M。采用競爭洗脫法研究了這兩個成分在β2-AR上的作用位點,證明兩個成分與β2-腎上腺素受體作用的位點是第三疏水區(qū)的天冬氨酸113,為復(fù)雜樣品藥物活性成分的篩選提供了方法,對高效創(chuàng)新藥物的開發(fā)具有重要意義。
[Abstract]:Study on the interaction of functional protein and small molecule ligands plays an important role in the complex signal involved reveal the life activities of transfer and development of efficient and innovative drugs. Biosensor with immobilized protein functions as the core of the kit, enzyme reactor and affinity color spectrum method is the main way to achieve the above research. However, due to protein the orientation and conformation of Immobilized Mixed interface, making it in the functional interactions between protein and drug research results deviate from the physiological state. Therefore, the research orientation and conformation control method of immobilized protein function has become one of the frontier research in medicine, chemistry and biology fields. Author's laboratory in the early work the establishment of the beta adrenergic receptor, 2- method, prove the method can be used to study the interaction of drug and receptor and drug. Component selection. Based on this, this thesis focuses on the stationary phase surface receptor orientation and conformation of a messy problem on the orientation and conformation of immobilized beta adrenergic receptors induced by 2-, a systematic study of the establishment and application of control methods. The study is expected to establish other immobilized protein functions as the core of the high sensitive method to provide the basis, have important significance to development of efficient and innovative drugs. This thesis consists of 4 chapters, the author's main contributions are as follows: (1) the establishment of a new beta adrenergic receptor 2- directed immobilization method of reversible, constructs the beta 2- adrenergic receptor orientation and conformation of the regulation model. The use of nickel ion and histidine tag beta 2- adrenergic receptor structure contained in the specific action between high density imidazole, directed the receptor reversible immobilized on macroporous silica surface was prepared by directional immobilization of beta 2- kidney Adrenoceptor chromatographic stationary phase. The results showed that the immobilized receptor can be got by this method. The unity of drugs clorprenaline hydrochloride as ligand, receptor of chromatography model specificity and stability were investigated. On this basis, the ephedrine hydrochloride and pseudoephedrine hydrochloride as conformation dependent probe, by changing the column temperature, mobile phase composition and conformation of ligand conditions on the immobilized receptor regulation. The results show that when the column temperature is 25 degrees centigrade, the most stable conformation can be immobilized receptor: when the mobile phase of phosphate buffer solution concentration of 5.0mmol/L (pH=7.40), immobilized beta 2- adrenergic receptor conformation optimal; in (S) - propranolol as ligand, the effects of drugs on the immobilized ligand 2- beta adrenergic receptor conformation, that the drug can induce ligand immobilized receptor conformational change, and strong The degree of 5.0nmol/L (S) - propranolol can make the immobilized beta 2- adrenergic receptor to obtain optimal conformation. This study regulation for other immobilized protein function orientation and conformation provides methodological reference. (2) application of stable conformation of beta adrenergic receptor 2- model to investigate the interaction of receptors with sulfuric acid Tebu He Lin and other seven kinds of drug ligand, provide the basis for elucidating the mechanism of drugs. The determination of terbutaline sulfate by affinity chromatography, methoxyphenamine hydrochloride, clorprenaline hydrochloride, tulobuterol hydrochloride, salbutamol sulfate, the binding constants of clenbuterol and bambuterol hydrochloride and 2- beta adrenergic receptor interaction and the number of binding sites. The results show that there are seven kinds of binding sites for drug 2- and beta adrenergic receptor binding constants are as follows: 1.84x104 104 M-1,1.71 * M-1,2.3 * 1O4M-1,2.09 * 104M -1,1.98 * M-1,2.37 104 * 104M-1 and 9.43 * 104 M-1; the number of binding sites is as follows: 8.3 * 10-4M, 3.88 * 10-5M, 1.02 * 10-5M, 2.16 * 10-5M, 7.5 * 10-5M, 1.82 * 10-5M, 2.47 * 10-5M. using microdialysis of the above results are verified, show that the two methods combined with constant and the number of binding sites in the same order of magnitude, that beta adrenergic receptor 2- chromatography model can be used to study the interaction of drug molecules and receptors for protein analysis, online drug interactions, high-throughput provides methodological reference. (3) application of stable conformation of 2- beta adrenaline receptor chromatography models were screened for Corydalis and 100 in the active ingredient, the mechanism by frontal chromatography and competitive displacement chromatography of the active ingredient and the receptor are discussed. The heating reflux preparation of corydalis and Stemona extract, receptor chromatography analysis and collection Liucheng divided by reversed-phase liquid chromatography tandem mass spectrometry were identified. The results showed that four of tetrahydroprotoberberines, protopine, Corydaline and four hydrogen columbamine as active ingredients in Rhizoma Corydalis and beta 2- adrenergic receptor specific action; stemoninine for 100 active ingredients in beta 2- adrenergic receptor. Further by frontal chromatography measured four hydrogen binding constants of berberine and protopine and beta 2-AR were 9.04 * 104M-1 and 4.30 * 104 M-1, the number of binding sites were binding sites of 6.67 * 10-4M and 5.88 * 10-4M. by competitive research of these two components in the beta 2-AR elution method two, prove the composition and effect of beta 2- adrenergic receptor sites is the hydrophobic region of third aspartic acid 113, provides a method of screening for complex samples of active pharmaceutical ingredients, has important significance to development of efficient and innovative drugs.

【學(xué)位授予單位】：西北大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R914

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