【正文】 剪接的內含肽基因進行融合表達，其中內含肽是在生物裂解過程中必要的工具。(A) A synthetic monomer gene is inserted into a cloning vector. (B) The gene is designed to contain recognition sites for two different restriction endonucleases， RE1 and RE2（ pflMΙ and BglΙ respectively）， at each of the coding sequence. (C)An insert is prepared by digestion of the vector with both RE1 and RE2 and subsequently ligated into the vector that has been linearized by digestion with only RE1. (D) The product contains two headtotail repeats of the original gene， with the RE1 and RE2 sites maintained only at the ends of the gene. (E) Additional rounds of RDL proceed identically， using products from previous rounds as starting materials[13]. 在純化蛋白方面的應用ELPs的應用目前研究最多的是在純化蛋白質方面。（B）ELPs單體基因序列的兩端設計成可被兩個限制性內切酶RE1與RE2（即分別為pflM Ι與BglΙ）識別的序列?；蛑亟M合成法更是由于RDL(recursive directional ligation，RDL)技術[13]的使用而被推向新的高度，其原理如圖1所示。ELPs的重復序列次數(shù)、濃度、Xaa、鹽濃度、pH及分子量是影響類彈性蛋白主要因素，由于 ELPs是一種對環(huán)境有很大敏感性的生物高分子，影響類彈性蛋白還有其它多種因素。因而，如果 ELPs鏈比較短時，就降低了聚合物鏈內部疏水性相互作用的效率，所以絮集作用就受抑制。但有時一些鹽離子可以提高，例如胍鹽[15]。C，這是因為 ELPs–[KV2F]家族的賴氨酸含量比 ELPs–[KV7F]家族高 2倍以上。Meyer D E等[14]分析 ELP[V5A2G3]文庫的與 ELPs濃度關系時，結果表明：隨著 ELPs濃度的上升而下降，并且呈線性關系。在相變發(fā)生時，增加溫度會使在水環(huán)境中的聚合物有序性得到加強，而包圍在疏水性殘基的有序水分子變成無序狀態(tài)，此時疏水性基團將與水溶液相分離，結果在整個變化過程中是趨于無序性的，這與熱力學第二定律相吻合[1011]。從構象變化上，Urry認為（VPGVG）重復單元在ProGly處結構為β轉角（β turn），而重復的β轉角形成右手螺旋的β螺旋（righthanded helix termedβspiral），而β螺旋可用來解釋效應與△機理（effect與△ mechanism）；在分子水平上，當溫度高于時，部分較為伸展且無序的構象轉變成較為緊湊的β螺旋[56]。其結構主要由五肽重復序列單元構成，即（ValProGlyXaaGly, VPGXG），源自于彈性蛋白的疏水區(qū)域，其中 Xaa可以是除 Pro以外任一氨基酸[1]。關鍵詞：類彈性蛋白多肽，相變溫度，非色譜純化，分子動力學模擬，相變機理AbstractElastinlike 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 () 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 network (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 ELP[KV8F20] had undergone phase transition at the 375K and suffered from denaturalization at 400 K when the ELP[KV8F20] was in inaqueous solutions。通過分析，推斷疏水作用與結合水的減少在ELP[KV8F20]的相變過程中起到關鍵作用。通過分子動力學模擬探討類彈性蛋白ELPs發(fā)生可逆相變 (ITT， inverse temperature transition)的分子機理及預測ELP[KV8F20]的相變溫度。將偽氨基酸組成中的的相關系數(shù)部分作為類彈性蛋白的特征向量，從類彈性蛋白序列出發(fā)，利用最小中位方差回歸，找出其序列相關系數(shù)的最佳階數(shù)。本人依法享有和承擔由此論文所產(chǎn)生的權利和責任。論文寫作中不包含其他人已經(jīng)發(fā)表或撰寫過的研究內容，如參考他人或集體的科研成果，均在論文中以明確的方式說明。ELPs的相變溫度是定量描述其相變溫度性質最便利的參數(shù)，通過相變溫度實測值探討與其序列組成、每一單體的Xaa數(shù)量、濃度等因素間的關系具有重要意義。M ELP[KV8F20]相變溫度降低至19℃，此ELPs序列有望開發(fā)成一新型純化標簽，為今后重組蛋白的非色譜分離純化奠定基礎。模擬過程中，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時發(fā)生相變，400K時ELPs已經(jīng)變性；在水溶液條件下，ELP[KV8F20]在350K時發(fā)生相變，375K時ELPs已經(jīng)變性?？偨Y了相關結論，這可為今后利用ELPs純化重組蛋白提供經(jīng)驗積累和理論依據(jù)。transition principle摘要 IABSTRACT II第一章 緒論 1 1 1 1 2 4 5 8 9第二章 使用偽氨基酸組成和BP神經(jīng)網(wǎng)絡預測類彈性蛋白多肽的相變溫度 10 10 10 10 12 氨基酸相關系數(shù)的階數(shù)的選擇 12 13 15 16第三章 類彈性蛋白多肽的從頭設計、非色譜純化及鹽效應研究 17 ELPS[KV8F20]的表達策略 17 ELPs的命名 17 17[KV8F20]的構建 18 18 18 1DNA Marker和試劑盒 19 19 20 20 21 ELP[KV8F20]的克隆 21 21 ELPs[KV8F20]的表達與純化 23 26 pUC19ELPs的酶切及ELPs基因片段回收 26 ELP[KV8F20]的誘導表達與純化 27 ELP[KV8F20]相變溫度的影響因素考察 27 31第四章 類彈性蛋白多肽的分子動力學模擬 32 NAMD模擬方法 32 NAMD軟件 32 32 33 33 ELPs[KV8F20]的MD模擬的建立 34 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 45第五章 結論 46第六章 研究展望 47參考文獻 48攻讀碩士學位期間發(fā)表學術論文情況 52致謝 53附圖 54 V第一章 緒論類彈性蛋白多肽(Elastinlike polypeptides，ELPs)是一種具有彈性功能且對環(huán)境溫度非常敏感的人造基因工程蛋白質聚合物。利用類彈性蛋白的可逆相變特性,使其在蛋白純化、作為藥物載體、組織工程等方面得到廣泛的應用[2]相變過程是 ELPs在水溶液中絮聚的過程，伴有伸展型多肽鏈的瓦解并與溶液相分離[34]。在(GVGVP)n疏水區(qū)域內有序體運動鏈節(jié)會產(chǎn)生相互作用，由于疏水區(qū)域內部運動鏈的阻尼作用，疏水性區(qū)域聚集會引起振動熵的上升，疏水性區(qū)域聚集的發(fā)生是伴隨著蛋白疏水部位共振，而溫度在一定范圍內上升時有助于疏水作用加強。當五肽重復序列次數(shù)較少時，ELPs的隨重復序列的重復次數(shù)的增加而下降較明顯，當五肽重復序列次數(shù)較大時，ELPs的隨重復序列重復次數(shù)的增加，其值變化不大。通常情況下，相似分子量的 ELPs–[KV2F]家族的比 ELPs –[KV7F]家族要高 16