本文選題：絲素蛋白　切入點：多肽　出處：《太原理工大學》2017年碩士論文

【摘要】：家蠶絲素蛋白是一類有著獨特的氨基酸組成和結晶結構的動物蛋白,因具有良好的生物相容性、優(yōu)異的機械性能、無毒和可生物降解等特點而廣泛的應用于生物醫(yī)學、光學和組織工程等領域。以再生絲素蛋白或重組絲蛋白溶液作為原材料,通過仿生的綠色化學方法獲取高性能、多功能的絲基材料是實現(xiàn)絲素蛋白應用的基礎。絲素蛋白在水溶液中的自組裝機理對于深入了解其結構與功能之間的關系具有十分重要的指導意義。目前對絲素蛋白在水溶液中的組裝過程和機制沒有統(tǒng)一和清晰的認識,采用的研究方法也難以方便直觀地監(jiān)測自組裝的過程。金納米粒子具有與其形狀、粒徑和聚集程度相關的表面等離子共振效應,金納米粒子發(fā)生聚集時會引發(fā)顆粒間的表面等離子耦合,表現(xiàn)出肉眼可見的紅色到紫色或藍色的變化。這種效應的應用逐漸發(fā)展成為以金納米粒子為探針的比色檢測方法,并應用于核酸、小分子和蛋白質等物質的比色檢測。為此,本文提出了以金納米粒子為探針研究絲素蛋白水溶液中組裝行為的新思路。在家蠶絲素蛋白的整個序列中,高度重復的六肽單元GAGAGS能夠形成反平行的β-折疊結構,進而構成絲蛋白的結晶區(qū)域,而另外一種六肽單元GAGAGY則有可能構成了其非結晶區(qū)。近年來以絲素蛋白序列中某些具有代表性的多肽序列作為簡化模型來研究絲素蛋白的組裝機理成為一種趨勢。本文選用十四肽(GAGSGAGAGSGAGY,GY-14)和八肽(GAGAGAGY,GY-8)分別作為絲素蛋白晶區(qū)與非晶區(qū)的簡化模型,并以多肽末端的酪氨酸原位還原的金納米粒子作為固定在多肽模型上的探針。通過溶液顏色以及紫外吸收峰的改變,研究外源性因素對多肽溶液組裝行為的影響規(guī)律,建立以金納米粒子為探針研究多肽在水溶液中組裝行為的方法。本文開展了以下研究:(1)金納米粒子原位制備條件與性能之間關系的研究:分別以多肽GY-8和GY-14作為還原劑,原位還原氯金酸合成了穩(wěn)定分散的球形金納米粒子,并研究了多肽與氯金酸的配比、溶液p H和溫度等不同制備條件對金納米粒子形態(tài)、分布以及穩(wěn)定性的影響。通過紫外-可見吸收光譜和透射電鏡表征確定了最佳的制備條件:以多肽GY-8為還原劑時,多肽與氯金酸摩爾比為5:2,p H為9,溫度為40℃;以多肽GY-14為還原劑時,多肽與氯金酸摩爾比為1:1,p H為7,溫度為35℃。(2)以金納米粒子為探針對多肽水溶液組裝行為的研究。分別研究了多肽GY-14在p H、金屬離子濃度以及溶劑組成等外源性因素影響下的自組裝行為,并應用紫外光譜、紅外光譜、透射電鏡和原子力顯微鏡等方法對組裝規(guī)律以及組裝結構進行了表征。結果表明:多肽GY-14在p H 4的酸性水溶液中構象由無規(guī)卷曲轉變?yōu)棣?折疊,溶液由紅色變?yōu)樽仙?紫外吸收峰紅移。Ca2+的引入會誘導多肽構象發(fā)生轉變,當離子濃度為5 m M和10 m M時,多肽自組裝形成由粒子連接而成的串珠狀的β-折疊結構,溶液分別由紅色變?yōu)樽仙退{色,吸收峰紅移。異丙醇溶液的加入使得多肽從無規(guī)卷曲構象轉變?yōu)棣?折疊構象,同樣形成串珠狀組裝體,溶液中出現(xiàn)紫色絮狀物并在短時間內沉淀,紫外吸收峰基本消失。本文以金納米粒子為探針,將多肽GY-14在水溶液中的組裝行為通過溶液顏色的改變以及紫外吸收峰的移動表現(xiàn)出來,為研究多肽的溶液組裝行為提供了一種簡單直觀的新途徑,為進一步研究絲素蛋白構象轉變機制和過程提供指導。
[Abstract]:Silk fibroin is a kind of a unique amino acid composition and crystal structure of animal protein, with good biocompatibility, excellent mechanical properties, non-toxic and biodegradable characteristics widely used in biomedical, optical and tissue engineering and other fields. The regenerated silk fibroin or recombinant silk protein solution as a raw material, to obtain high performance by the green chemistry method of bionic, multifunctional material filaments is the basis for the application of fibroin protein. The silk fibroin in aqueous solution of the self-assembly mechanism has a very important significance for understanding the relationship between its structure and function. The silk fibroin assembly in water in the solution process and the mechanism is not clear and unified understanding, the research methods are also difficult to conveniently and visually monitoring a process of self-assembly. The gold nanoparticles have its shape, size and The surface plasmon resonance effect is related to the degree of aggregation and aggregation of gold nanoparticles leads to surface plasmon coupling between particles, exhibit visible red to purple or blue. The application of this effect has gradually become the gold nanoparticle colorimetric method for measuring probe, and applied to the nucleic acid, low molecular weight and protein the colorimetric detection is proposed in this paper. Therefore, using gold nanoparticles as a new idea of assembling behavior of silk fibroin solution probe in water. In the entire sequence of silk fibroin, height six peptide unit GAGAGS repeats to form antiparallel beta folding structure, and thus constitute the crystalline regions of silk, and another six GAGAGY peptide unit is likely to constitute the non crystalline region. In the recent years on the silk fibroin peptide sequence of protein sequences in some representative as a simplified model to The assembly mechanism of silk fibroin has become a trend. This paper selected fourteen peptides (GAGSGAGAGSGAGY, GY-14) and eight (GAGAGAGY, GY-8) peptides were used as the silk fibroin crystalline region and amorphous region of the simplified model, and the in situ reduction of tyrosine peptide gold nanoparticles as probes immobilized on the polypeptide model. The color of the solution. And the ultraviolet absorption peak changes, the influence of exogenous factors on the peptide solution assembly behavior, build with gold nanoparticle assembly of peptide probe method in aqueous solution. In this paper, we conducted the following research: (1) study on the relationship between gold nanoparticles in situ preparation conditions and performance respectively with polypeptides GY-8 and GY-14 as the reducing agent, in situ reduction of chloroauric acid stable dispersion of spherical gold nanoparticles were synthesized, and studied the ratio of peptides with chloroauric acid solution, P H and different temperature etc. The morphology of gold nanoparticles preparation conditions, distribution and stability. The influence by UV Vis absorption spectroscopy and transmission electron microscopy characterization of the best preparation condition was determined: the peptide GY-8 as reducing agent, polypeptide and chloroauric acid molar ratio of 5:2, P and H was 9, the temperature of 40 DEG GY-14 for reduction in polypeptide; agent, polypeptide and chloroauric acid molar ratio of 1:1, P and H was 7, the temperature of 35 DEG. (2) research on probe polypeptide assembly behavior in aqueous solution by using gold nanoparticles as GY-14 in P H. The peptide self-assembly behavior were studied, the effect of metal ions concentration and solvent composition under the exogenous factors then, using ultraviolet spectrum, infrared spectroscopy, transmission electron microscopy and atomic force microscopy were used to characterize the assembly rules and assembly structure. The results show that the conformation of peptide GY-14 in P H 4 acid solution by random coil transition folding beta, the solution from red to purple The introduction of color, UV absorption peak red shift.Ca2+ will induce peptide conformational change when the ion concentration of 5 m M and 10 m M, peptide self-assembly formed by particles formed by connecting the beaded beta folding structure, solution was changed from red to purple and blue absorption peak red shift. Isopropanol the solution of adding polypeptides from random coil conformation into p-sheet conformation, the same form of beaded assembly, solution purple floc and precipitation in a short period of time, the ultraviolet absorption peak disappeared. In this paper, gold nanoparticles as probe, the assembly of peptide GY-14 in aqueous solution by the color of the solution change and UV absorption peak of mobile performance, provides a new way for the solution of a simple polypeptide assembly, to provide guidance for further research on the conformation transition of silk fibroin mechanism and process.

【學位授予單位】：太原理工大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：TB383.1;O629.7

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