【作者】 王泽方；

【导师】 乔明强；

【作者基本信息】 南开大学 ， 微生物学， 2010， 博士

【摘要】 疏水蛋白是丝状真菌产生的小分子量蛋白质,其最大特点是能够在界面形成一层双亲性的蛋白膜以此来反转所覆盖界面的性质。自组装的疏水蛋白应用非常广泛,例如在乳化、蛋白分离技术、生物传感器和生物芯片等领域。在前期研究中,本研究室从真菌灰树花平皿菌丝中发现了一个新的疏水蛋白HGFI。随着对HGFI蛋白研究的不断深入,遇到以下三个问题：一是HGFI蛋白的大规模生产和纯化问题；二是需要发展HGFI蛋白应用的新方向；三是HGFI蛋白的自组装及固定外源生物分子的机制不明。这三个问题也是疏水蛋白研究领域所面临的共同问题。在本文的第二章到第六章将系统研究以上三个问题。在第二章中,进行了HGFI在大肠杆菌中的表达纯化与性质研究工作。hgfI基因被克隆到了pET-28a表达载体中,随后被转化进入大肠杆菌BL21菌株SDS-PAGE电泳显示,经过优化诱导所用IPTG的用量和时间之后,重组HGFI蛋白主要以包涵体形式表达。随后利用电洗脱技术对包涵体中HGFI进行了纯化。接触角测量表明,经过复性的重组HGFI蛋白具有部分自组装活性。纯化后的HGFI被用于生产多克隆抗体,ELISA和Western结果显示HGFI的多克隆抗体不仅能够识别大肠杆菌表达的重组HGFI,还可以识别产自灰树花菌丝的天然HGFI。最后利用免疫荧光技术对HGFI蛋白在灰树花菌丝进行了定位,结果显示HGFI不但出现在灰树花菌丝的细胞壁上,还出现在了菌丝出芽的位置。该结果表明HGFI蛋白在灰树花的菌丝细胞壁形成和出芽过程中起了重要作用。在第三章中,进行了HGFI在毕氏酵母中的表达纯化与性质研究工作。hgfI基因被克隆到了pPIC9表达载体中,随后被转化进入了毕氏酵母GS115菌株。SDS-PAGE、Western以及MALDI-TOF质谱结果显示大小在8 KDa左右的重组HGFI被分泌到了培养基中。通过控制甲醇的浓度和诱导时间,HGFI蛋白的表达条件得到了优化。超滤和高压液相色谱被用来纯化HGFI蛋白。经过以上两个步骤的纯化,每升酵母发酵液能得到86mg的HGFI蛋白。X射线光电子能谱和接触角测量实验表明重组HGFI可以在疏水的硅烷化玻璃、特氟龙以及亲水的云母表面自组装成膜。细胞毒性测试表明自组装的HGFI蛋白膜具有良好的生物相容性,可以帮助人主动脉平滑肌细胞在组织工程材料-聚己内酯上更好的增殖。在第四章中,进行了HGFI分散多壁碳纳米管及固定抗体分子的应用研究。经过30 min的超声,多壁碳纳米管能够分散在0.1 mg/mL的HGFI溶液中。光学吸收和透射电镜实验表明多壁碳纳米管的分散溶液很稳定。X射线光电子能谱、红外光谱、拉曼光谱和热重分析实验表明了HGFI能够通过疏水作用力,非共价地吸附在多壁碳纳米管表面,使其表面呈现亲水性。石英晶体微天平和免疫分析实验表面了HGFI修饰的多壁碳纳米管可以用来在溶液中固定免疫球蛋白G。在第五章中,进行了利用亲水带电的HGFI膜修饰聚苯乙烯表面及其在时间分辨免疫荧光分析中的应用研究。石英晶体微天平实验表明在pH 5时,经过两个吸附阶段,疏水蛋白HGFI可以在聚苯乙烯表明形成一层带负电的亲水单层膜。X射线光电子能谱和接触角测量实验表明自组装的疏水蛋白可以使得聚苯乙烯表面在3个月内保持亲水性。原子力显微镜实验表明,聚苯乙烯表面的粗糙度在HGFI吸附后变小,抗体分子以“直立”的形式在亲水的疏水蛋白膜上形成了致密的单层膜。而在疏水的聚苯乙烯表面上,抗体分子以“侧立”形式形成了疏松的多层膜。时间分辨免疫荧光分析显示在HGFI修饰的聚苯乙烯表面,癌胚抗原检测的线性范围为15-600 ng/mL。不同检测批次和批间的标准偏差在4%以内。以上结果表明时间分辨免疫荧光分析在HGFI修饰过聚苯乙烯表面具有很高的灵敏性。在第六章中,通过石英晶体微天平技术系统地研究了在不同pH和离子强度条件下,两种疏水蛋白在巯醇表面的自组装以及疏水蛋白固定外源生物分子的机制。实验结果表明：第一,Ⅰ型疏水蛋白和Ⅱ型疏水蛋白的自组装过程和结果都不同。Ⅰ型蛋白的自组装受静电力影响较大,在巯醇表面主要形成“弹性膜”。Ⅱ型蛋白的自组装受静电力影响较小,在巯醇表面主要形成“刚性膜”。第二,外源蛋白在疏水蛋白表面的固定化主要依靠静电力驱动。通过改变pH和离子强度,可以有效控制外源蛋白在疏水蛋白膜上的吸附。随后的研究利用了以上结论制备了基于疏水蛋白的用于检测C-反应蛋白的压电型免疫传感器。通过以上的研究工作,解决了疏水蛋白的大规模生产和纯化问题,指出了疏水蛋白应用的新方向,并且阐明了疏水蛋白自组装和固定化的机制。本文的研究工作为更加深入的研究疏水蛋白提供了物质和理论基础。更多还原

【Abstract】 Hydrophobins are small proteins that are produced by filamentous fungi. The most important feature of hydrophobins is that they can form an amphipathic membrane of 10-nm-thick, reversing properties of the interface coated by them. The self-assembly of hydrophobins is interesting for many applications, which include personal care and emulsions, separation technologies, biosensors and biochips. Recently, our laboratory identified a new hydrophobin HGFI from Grifola frondosa (G. frondosa). With the deepening of research on HGFI, we encountered three major problems. The first one is that it is difficult to produce and purify HGFI in a large scale; the second one is that it is urgently to develop new application areas of HGFI; the last one is that the self-assembly property and immobilization mechanism of HGFI have not been determined yet. The aims of this work are to shed light on the above three problems which are also common ones existing in the whole hydrophobin family.In chapterⅡ, the expression, purification and characterization of a recombinant HGFI were described. The hgfI gene was cloned into pET-28a expression plasmid and transformed into Escherichia coli (E. coli) BL21 strain. SDS-PAGE analysis showed that recombinant HGFI was satisfactorily expressed by optimizing the concentration and induction time of IPTG and purified by electroelution. Water contact angle measurement indicated that the biological activity of purified HGFI was partially preserved after refolding. Then the purified HGFI was used to immunize adult rabbits to produce antiserum. ELISA and Western blot analysis indicated that the produced antiserum could detect both HGFI protein expressed in prokaryotic cells (E. coli) and in eukaryotic cells (G. frondosa). Furthermore, the antiserum was used to determine localization of HGFI in G. frondosa cells by an immunofluorescence technique. The results demonstrated that HGFI protein was localized in the cell wall, especially at the budding position of hypha, suggesting that HGFI plays important roles in the cell wall formation and budding process of G. frondosa.In chapter III, the recombinant HGFI (rHGFI) was successfully expressed by using pPIC9 vector in Pichia pastoris. SDS-PAGE and Western blotting demonstrated that rHGFI, an 8 kDa protein, was secreted into the culture medium. The culture conditions of the transformant strain were optimized by controlling the methanol concentration and induction time. Ultrafiltration and reverse-phase high performance liquid chromatography were used to perform a large-scale purification of rHGFI. A stable production of rHGFI around 86 mg/L was achieved after the two-step purification. X-ray photoelectron spectroscopy and water contact angle measurements indicated that the functional rHGFI could self-assemble on hydrophobic siliconized glass and Teflon, as well as on hydrophilic mica surfaces. A methylthiazol tetrazolium assay showed that rHGFI film could facilitate human aortic smooth muscle cell proliferation on polycaprolactone due to its cytocompatibility.In chapter IV, HGFI protein was used to disperse multi-walled carbon nanotubes (MWCNTs) and immobilize immunoglobulin G in water. MWCNTs could be effectively dispersed by 30-min sonication in a 0.1mg/mL HGFI solution. Optical absorption and transmission electron microscopy provide evidence for individually stable dispersed MWCNTs. X-ray photoelectron, Fourier transform infrared, and Raman spectroscopies as well as thermogravimetric analysis suggested that HGFI can non-covalently bind to MWCNTs through hydrophobic interaction, rendering them hydrophilic. A quartz crystal microbalance and immunological sandwich assay were used to demonstrate that the HGFI-coated MWCNTs can be used to immobilize human immunoglobulin G in solution.In chapter V, a hydrophilic and charged hydrophobin HGFI film on the polystyrene surface was used as a solid support for immobilizing antibodies in time-resolved immunofluorometric assay (TR-IFMA). Quartz crystal microbalance with dissipative monitoring revealed that hydrophobin could form an intact negatively charged monolayer on the polystyrene undergoing two adsorption phases at pH 5. X-ray photoelectron spectroscopy and water contact angle measurements showed that the self-assembly hydrophobin on polystyrene can render its surface very hydrophilic for three months. Atomic force microscope indicated that the roughness of the polystyrene was reduced after modification. Moreover, an integrated antibody monolayer was "end-on" adsorbed on the hydrophilic hydrophobin film rather than multilayers on the unmodified polystyrene in a "side-on" orientation. TR-IFMA showed that a linear calibration curve was obtained in the concentration range from 15-600 ng/mL of and the relative standard deviation was less than 4% on the hydrophobin-modified polystyrene which showed higher sensitivity than unmodified polystyrene in the TR-IFMA.In the last chapter, both how hydrophobins self-assemble on the hydrophobic thioled-surface and how proteins adsorb onto hydrophobins were systematically studied. The results showed that the electrostatic force can affect the self-assembling of Class I hydrophobins more than that of Class II ones. Furthermore, Class I hydrophobins mainly form a soft layer on the thioled-surface, rather than a rigid one formed by class II hydrophobins. It was also found that the surface adhesion of hydrophobins was due to electrostatic interactions. By controlling solution conditions such as pH and ionic strength, several types of proteins readily adsorb onto hydrophobins. The usability of this type of adhesion was demonstrated by making a simple quantitative immunological sandwich assay.In conclusion, this study solved the problem of large-scale production and purification of hydrophobins and given two new application directions of hydrophobin, as well as ultimately revealed the self-assembly and immobilization mechanism of hydrophobins. This study can provide a practical and theoretical foundation for more in-depth studies on hydrophobins.更多还原