【正文】 ........ 33 ELPs[KV8F20]的 MD 模擬的建立 ........................................................................ 34 動(dòng)力學(xué)模擬的結(jié)果與分析 ......................................................................................... 36 RMSD 值分析 .......................................................................................................... 36 氫鍵數(shù)目分析 .......................................................................................................... 38 末端距值分析 .......................................................................................................... 42 溶劑可及性表面積分析 ........................................................................................ 43 本章小結(jié) ...................................................................................................................... 45 第五章 結(jié)論 ...................................................................................................................... 46 第六章 研究展望 ............................................................................................................. 47 參考文獻(xiàn) ............................................................................................................................ 48 攻讀碩士學(xué)位期間發(fā)表學(xué)術(shù)論文情況 ........................................................................... 52 致謝 .................................................................................................................................... 53 附圖 .................................................................................................................................... 54 華僑大學(xué)碩士研究生畢業(yè)論文 1 第一章 緒論 類(lèi)彈性蛋白多肽的概述 類(lèi)彈性蛋白多肽簡(jiǎn)介 類(lèi)彈性蛋白多肽 (Elastinlike polypeptides， ELPs)是一種具有彈性功能且對(duì)環(huán)境溫度非常敏感的人造基因工程蛋白質(zhì)聚合物。模擬過(guò)程中， ELP[KV8F20] 在水溶液與 1M 的 NaCl 溶液均發(fā)生疏水縮聚， 推斷在水溶液條件下， ELP[KV8F20]在 375K 時(shí)發(fā)生相變， 400K 時(shí) ELPs 已經(jīng)變性；在水溶液條件下， ELP[KV8F20]在 350K 時(shí)發(fā)生相變， 375K 時(shí) ELPs 已經(jīng)變性。 ELPs 的相變溫度是定量描述其相變溫度性質(zhì)最便利的參數(shù)，通過(guò)相變溫度實(shí)測(cè)值探討與其序列組成、每一單體的 Xaa 數(shù)量、濃度等因素間的關(guān)系具有重要意義。本人依法享有和承擔(dān)由此論文所產(chǎn)生的權(quán)利和責(zé)任。 通過(guò)分子動(dòng)力學(xué)模擬探討類(lèi)彈性蛋白 ELPs 發(fā)生可逆相變 (ITT， inverse temperature transition)的分子機(jī)理及預(yù)測(cè) ELP[KV8F20]的相變溫度。 關(guān)鍵詞： 類(lèi)彈性蛋白多肽，相變溫度，非色譜純化，分子動(dòng)力學(xué)模擬，相變機(jī)理 華僑大學(xué)碩士研究生畢業(yè)論文 II Abstract Elastinlike polypeptides (Elastinlike polypeptides, ELPs) are elasticity and environmentally responsive biopolymers. Due to their reversible phase transition characteristic, Elastinlike polypeptides (ELPs) have been used for a number of applications, such as protein purification, drug delivery, and tissue engineering. For the ELPs, the transition temperature ( tT ) is the most convenient parameters for quantificational description of the ELP properties. It is of great importance to explore the relationship between the transition temperature and the sequence characteristics, the number of Xaa of each monomer and the concentration of ELP. The best order of the correlation coefficient for pseudoamino acid position was obtained by using Least Median of Squares Regression from sequence. The uniform design was used to optimize the running parameters and leaveone out crossvalidation was carried out to evaluate the model of back propagation neural work (BPNN) and support vector machines, respectively. The results showed that the predicted model obtained by BPNN was the best, of which the mean absolute error and root mean squared error was ℃ and ℃ , respectively. Then, we de novo designed a novel ELPs gene and cloned it into the modified expression vector pET22b. Then, we transformed the rebinant expression vector pET22bELPs into Escherichia coli BL21(DE3). Upon induction by Isopropyl βDThiogalactoside (IPTG), ELPs was expressed and purified by a nonchromatographic purification method named inverse temperature cycling. The influences of salts types and concentrations on ELPs were also determined. The results showed that the transition temperature of the ELP[KV8F20] decreased to 19℃ by mmol/L Na2CO3. Due to its small molecular weight and sensitivity to salt, the ELPs might be a useful purification tag, which can provide a reliable and simple nonchromatographic method for purification of the rebinant protein by inverse transition cycling. Finally， we concerned on the molecular mechanism for ITT(Inverse temperature transition) of ELPs(elastinlike peptides)， and prediction for the transition temperature of ELP[KV8F20]. As the temperature increased, ELPs undergoes changing from a extended to folded conformation. Molecular dynamic simulations were performed 9ns in explicit water and 1M NaCl solution for ELP[KV8F20] at five different temperature between 300 K and 400 K. During simulation processing, ELP[KV8F20] had hydrophobic collapse in explicit water and 1M NaCl solution. According to simulation result,we concluded that 華僑大學(xué)碩士研究生畢業(yè)論文 III ELP[KV8F20] had undergone phase transition at the 375K and suffered from denaturalization at 400 K when the ELP[KV8F20] was in inaqueous solutions。從構(gòu)象變化上， Urry認(rèn)為（ VPGVG）重復(fù)單元在 ProGly處結(jié)構(gòu)為 β轉(zhuǎn)角（ β turn），而重復(fù)的 β轉(zhuǎn)角形成右手螺旋的 β螺旋（ righthanded helix termedβspiral），而 β螺旋可用來(lái)解釋 tT 效應(yīng)與 △ tT 機(jī)理（ tT effect與 △ tT mechanism）；在分子水平上，當(dāng)溫度高于 tT 時(shí)，部分較為伸展且無(wú)序的構(gòu)象轉(zhuǎn)變成較為緊湊的 β螺旋 [56]。 Meyer D E等 [14]分析 ELP[V5A2G3]文庫(kù)的 tT 與 ELPs濃度關(guān)系時(shí)，結(jié)果表明： tT 隨著 ELPs濃度的上升而下降，并且呈線(xiàn)性關(guān)系。但有時(shí)一些鹽離子可以提高 tT ，例如胍鹽 [15]。 ELPs的重復(fù)序列次數(shù)、濃度、 Xaa、鹽濃度、 pH及分子量是影響類(lèi)彈性蛋白 tT 主要因素，由于 ELPs是一種對(duì)環(huán)境有很大敏感性的生物高分子，影響類(lèi)彈性蛋白 tT 還有其它多種因素。（ B） ELPs 單體基因序列的兩端設(shè)計(jì)成可被兩個(gè)限制性?xún)?nèi)切酶 RE1 與 RE2（即分別為 pflM Ι與 BglΙ）識(shí)別的序列。 Banki M R 等 [19]設(shè)計(jì)一種自我剪接的 ELPs，這種能自我剪接的 ELPs 由 ELPs 基因與能自我剪接的內(nèi)含肽基因進(jìn)行融合表達(dá)，其中內(nèi)含肽是在生物裂解過(guò)程中必要的工具。 作為生物材料的 ELPs 已廣泛當(dāng)作可再生及修繕的醫(yī)藥。 Urry 等通過(guò)實(shí)驗(yàn)表明植入 ELPs 后僅導(dǎo)致極微小的毒性與免疫反應(yīng) [22]。在三維結(jié)構(gòu)上小孔相互連接（ interconnected）的 ELPs 膠體可促進(jìn)纖維原細(xì)胞生長(zhǎng)，提高滲透性，利用該特性， ELPs 膠體還可應(yīng)用于外科整形。此外，ELPs 作為藥物載體，還可向動(dòng)關(guān)節(jié)、背根輸送藥物 [3839]。 MacKay 等 [43]設(shè)計(jì)出含 ELPs 的嵌合肽，此嵌合肽利用 ELPs 自組裝的特點(diǎn)可以自發(fā)形成納米微粒，通過(guò)老鼠的腫瘤模型實(shí)驗(yàn)，結(jié)果表明單純藥物運(yùn)輸相比（ free drug），嵌合肽耐劑量（ maximum tolerated）高四倍，且 注射一劑量（ single dose）后，腫瘤幾乎完全衰退。 Roger Rousseau 等 [