本文關(guān)鍵詞： 分子模擬 結(jié)合自由能 虛擬篩選 整合酶 艾滋病　出處：《成都學(xué)院》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：HIV-1是全球流行的傳染病——艾滋病的病原體,嚴(yán)重威脅著人類的健康,針對(duì)HIV-1相關(guān)蛋白的藥物設(shè)計(jì)成為抗艾滋病藥物研究的熱點(diǎn)領(lǐng)域。HIV-1相關(guān)的蛋白主要包括逆轉(zhuǎn)錄酶、整合酶和蛋白酶,它們分別在病毒的生命周期中發(fā)揮著不同的功能,因此,研究藥物與三個(gè)酶的作用機(jī)制對(duì)于后續(xù)抗艾滋病的藥物設(shè)計(jì)具有重要意義。本論文主要采用計(jì)算機(jī)輔助藥物設(shè)計(jì)方法對(duì)三個(gè)蛋白進(jìn)行了系列研究,內(nèi)容包括4個(gè)方面:沙奎那韋與HIV-1蛋白酶的分子識(shí)別;HIV-1逆轉(zhuǎn)錄酶的功能運(yùn)動(dòng)性分析;HIV-1整合酶抑制劑的篩選平臺(tái)及分子識(shí)別;HIV-1整合酶在大腸桿菌中的表達(dá)和純化。(1)通過(guò)分子動(dòng)力學(xué)模擬研究了SQV與蛋白酶的分子識(shí)別機(jī)理,并計(jì)算了二者形成的氫鍵以及重要氨基酸對(duì)識(shí)別的貢獻(xiàn)。計(jì)算得到的B因子與實(shí)驗(yàn)值具有顯著的相關(guān)性表明模擬過(guò)程是可靠的;SQV與Asp25'和Asp25之間穩(wěn)定的氫鍵是結(jié)合的重要作用力;Gly49'、Gly27'、Pro81和Asp29'對(duì)于SQV-蛋白酶識(shí)別的能量貢獻(xiàn)較大。(2)采用粗�；Ｐ头治隽四孓D(zhuǎn)錄酶各個(gè)功能區(qū)域的運(yùn)動(dòng)性,并采用分子對(duì)接預(yù)測(cè)了逆轉(zhuǎn)錄酶與NAD化合物的復(fù)合物模型。結(jié)果顯示,手指區(qū)與RNase H區(qū)的開合運(yùn)動(dòng)是逆轉(zhuǎn)錄酶最主要的功能性運(yùn)動(dòng),對(duì)于發(fā)揮逆轉(zhuǎn)錄功能具有重要的意義;分子對(duì)接表明反式雙鍵是NAD化合物與RT結(jié)合的優(yōu)勢(shì)構(gòu)象。(3)通過(guò)序列比對(duì)、虛擬篩選和結(jié)合自由能計(jì)算研究PFV IN-DNA體系作為IN抑制劑篩選平臺(tái)的潛力,并通過(guò)分子動(dòng)力學(xué)模擬及構(gòu)象分析研究了NRD化合物與IN的分子識(shí)別。結(jié)果表明,40.8%的序列相似度,26.43的篩選效率和結(jié)合自由能計(jì)算值與實(shí)驗(yàn)值的顯著相關(guān)性都證明了PFV IN-DNA作為虛擬篩選平臺(tái)的可靠性;DTG的結(jié)構(gòu)可以分為親水性和疏水性部分,其中2個(gè)Mg2+、3個(gè)水分子以及周圍的DDE保守氨基酸與親水性區(qū)域結(jié)合,而病毒DNA中的堿基以及Tyr212、Pro214等氨基酸則與疏水部分結(jié)合;構(gòu)象運(yùn)動(dòng)性分析表明,導(dǎo)致IN-DNA的運(yùn)動(dòng)性降低可能是IN抑制劑的抑制機(jī)理之一。(4)將含有F185K/C280S雙突變的pET28a-IN重組質(zhì)粒導(dǎo)入大腸桿菌細(xì)胞內(nèi)并表達(dá),并用親和層析及SDS-PAGE法純化蛋白。結(jié)果顯示,通過(guò)親和層析得在細(xì)胞破碎上清液中得到較多的整合酶蛋白質(zhì),表明所采用的實(shí)驗(yàn)方法是可靠的。
[Abstract]:HIV-1 is the pathogen of AIDS, which is a global infectious disease. It is a serious threat to human health. Drug design for HIV-1 related proteins has become a hot topic in the research of anti-AIDS drugs. HIV-1 related proteins mainly include reverse transcriptase. Integrase and protease, which play different roles in the virus's life cycle, therefore, It is important to study the action mechanism of drugs and three enzymes for the subsequent anti-AIDS drug design. In this paper, a series of studies on the three proteins were carried out by using the method of computer-aided drug design. The content includes four aspects: molecular recognition of sarquinavir and HIV-1 protease; functional mobility analysis of HIV-1 reverse transcriptase; screening platform for HIV-1 integrase inhibitor; expression and purification of HIV-1 integrase in Escherichia coli. The molecular recognition mechanism of SQV and protease was studied by molecular dynamics simulation. The hydrogen bond formed by them and the contribution of important amino acids to recognition are also calculated. The calculated B factor is significantly correlated with the experimental value, which indicates that the simulation process is reliable and the stable hydrogen bond between SQV and Asp25 'and Asp25 is bound. The important action force of Gly49A, Gly27P27 Pro81 and Asp29', was used to analyze the motility of various functional regions of reverse transcriptase by using coarse granulation model, which contributed significantly to the energy contribution of SQV- protease recognition. Molecular docking was used to predict the complex model of reverse transcriptase and NAD compounds. The results showed that the opening and closing movement between finger region and RNase H region was the most important functional movement of reverse transcriptase, which had important significance for exerting reverse transcriptase function. Molecular docking indicates that trans-double bond is the dominant conformation for the binding of NAD compounds to RT.) by sequence alignment, virtual screening and calculation of binding free energy, the potential of PFV IN-DNA system as an IN inhibitor screening platform is studied. Molecular dynamics simulation and conformation analysis were used to study the molecular recognition of NRD compounds and IN. The results showed that the screening efficiency of 40.8% sequence similarity and the significant correlation between the calculated value of binding free energy and the experimental value were proved. The structure of PFV IN-DNA as a virtual screening platform can be divided into hydrophilic and hydrophobic parts. Two Mg2, three water molecules and the surrounding DDE conserved amino acids bind to the hydrophilic region, while the bases in the viral DNA and the amino acids Tyr212OPro214 are partially bound to the hydrophobic region. The reduction of IN-DNA motility may be one of the inhibitory mechanisms of IN inhibitors. (4) the recombinant pET28a-IN plasmid containing double mutation of F185K / C280S was introduced into Escherichia coli cells and expressed. The protein was purified by affinity chromatography and SDS-PAGE. Through affinity chromatography, more integrase proteins were obtained in the supernatant of cell fragmentation, which indicated that the experimental method was reliable.
【學(xué)位授予單位】：成都學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R91

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