【作者】 李莉；

【导师】 胡远扬；

【作者基本信息】 武汉大学 ， 微生物学， 2003， 博士

【摘要】 长期以来人们一直在探索着生物大分子的结构与其功能之间的关系。一般情况下，只有知道生物大分子的原子水平结构才能更好地了解其功能。所以生物大分子高分辨结构的研究对我们进一步了解病毒的装配过程和病毒在感染过程中在细胞内的一系列活动都至关重要。 最早用于研究生物大分子高分辨结构的是X射线晶体学。可用于研究三维晶体的原子结构。但要求被研究的生物大分子能够生长成具有一定尺寸大小的三维晶体。由于这一局限，用X射线衍射技术研究不能结晶的生物大分子就很困难。 核磁共振波谱学也可很好地研究溶液中生物大分子的原子级结构及动态的分子间的相互作用，但由于大分子量的生物大分子复合体的共振谱异常复杂，难以解释。所以，利用这一技术处理的生物大分子，其分子量一般不超过35Kda。 用含水冷冻电子显微术进行生物大分子三维重构是近年来迅速发展的一门新兴学科。低温电子显微术是对含水生物大分子进行快速冷冻到液氮或液氦温度，并在低温条件下采用低电子剂量成像，从而可在高分辨水平上研究生物大分子的三维结构。而在传统电镜中，一般采用负染、化学固定、脱水等方法处理生物样品，使生物样品的高分辨结构信息失真，所以这种结构研究只局限于很低分辨率水平。而在低温电子显微术中，由于含水样品被冷冻的速率太快，样品内部的水来不及结晶而形成玻璃态的冰，这样整个生物大分子样品就被包埋在一薄层冰中。这层非晶态的薄冰一方面可作为支撑膜支撑样品，另一方面也可很好地在电镜的镜筒高真空中保存含水冷冻样品中的水，从而更好地保护含水生物大分子样品，使样品处于或接近于其生理活性状态，保持样品的高分辨结构信息。使生物大分子样品的高分辨三维结构研究成为现实。 由于细小病毒主要感染人和哺乳动物，导致许多疾病。所以对细小病毒的研究比较深入。目前对许多动物细小病毒的生物学特性都比较清楚，如基因组结构、病毒粒子的衣壳蛋白结构以及结构与功能之间的关系，特别是其中的犬细小病毒、猫肠炎细小病毒、和小鼠细小病毒的晶体结构已经在近原子水平分辨率上得到阐述。然而对浓核病毒的研究却仍然有限。本文试图通过对嶂螂浓核病毒三维结构的研究，来阐明这方面的不足。 素有活化石之称的嶂螂出现于3亿多年以前，是一种非常古老的昆虫。在我国分布最广的嶂螂种类是黑胸大蛾。黑胸大蜂浓核病毒是胡远扬等人1991年在国内首次报道并在国内外第一个正式分类鉴定的蜂螂浓核病毒。同时嶂螂也是一种危害不亚于蚊虫，苍蝇及老鼠的重要卫生害虫之一，传播许多疾病。该病毒属细小病毒科、浓核病毒亚科。它与其它细小病毒一样，病毒粒子无包膜，呈球状二十面体对称，直径22nm，基因组为单链线状DNA分子，含有五种结构蛋白，嶂螂浓核病毒基因组全长为5454个核昔酸，于C含量为38.13%。蜂螂浓核病毒可以感染嶂螂除中肠外的绝大部分组织。通常浓核病毒有很高的致病性，能在几天之内杀死大约90%的宿主幼虫，所以用浓核病毒作为杀虫剂很有潜力。因而希望进一步弄清楚它的结构和功能之间的关系。这对我们进一步了解病毒的浸染和增殖机理，有效地利用免疫学方法或基因工程方法改造昆虫浓核病毒，使之作为杀虫剂来控制和消灭有害昆虫都有极其重要的意义。 本论文对嶂螂浓核病毒的基因组结构与其它细小病毒和浓核病毒的基因组结构进行了对比分析，发现嶂螂浓核病毒的基因组结构以及编码蛋白都有自己独特的特点。一般细小病毒基因组有两个主要的开放阅读框，位于同一条DNA链上，左边O灯编码一到两种非结构蛋白伽sl和NSZ)，右边ORF编码三种结构蛋白(v卫l，VPZ和VP3)。其中主要成分是vPZ。而浓核病毒两条DNA链上都含有开放阅读框，右边含有一个开放阅读框(ORFI)编码所有的四种结构蛋白，左边含有三个开放阅读框(ORFZ，ORF3和ORF4)编码非结构蛋白。其中主要结构蛋白是V卫4。但蜂螂浓核病毒含有五种结构蛋白，有六个开 II放阅读框，正链有3个大的阅读框(o RFa，O侧叨，ORFy)，负链上也有3个大的阅读框(ORFI，ORFZ，ORF3)，阅读框都集中在每条链的右端，并且有基因重叠现象。通过嶂螂浓核病毒基因组编码蛋白与其它细小病毒同源性的比较推测ORFa编码非结构蛋白，而ORFI和ORFZ编码五种结构蛋白。 用SDS一PAGE电泳法对嶂螂浓核病毒的衣壳蛋白进行了分析，结果显示嶂螂浓核病毒衣壳蛋白由5条多肤组成，分子量分别为:VPI:52 kDa;VPZ:56 kDa;VP3: 79 kDa;VP4: 82 kDa;VPS:105 kDa。并利用凝胶成像系统分析软件对各蛋白质在衣壳中所占比例进行了计算，百分含量分别为:VPI:42.7%;VPZ:6.90%:VP3:31.9%;VP4:12.7%:VPS:5.75%。根据SDS一PAGE凝胶蛋白质条带密度以及百分含量计算结果确定VPI为蟀螂浓核病毒的主要结构蛋白。 本论文的重点是利用低温电子显微术和三维重构法对嶂螂浓核病毒的三维结构进行研究。我们获得了分辨率为23人的嶂螂浓核病毒的三维结构，研究表明嶂螂浓核病毒含有五种结构蛋白，60个蛋白亚基按T二1的对称二十面体结更多还原

【Abstract】 For a long time people have been trying to understand the relationship between the structure and function of the macromolecular complexes. In general, only with a good understanding of the structure of the macromolecular complexes at atomic resolution level, can its function be better understood. Therefore, investigations into the three-dimensional (3D) virus structures at a high resolution are crucial to the understanding of the fundamental processes of virus assembly and cellular events during viral infections.X-ray crystallography was the first method for structural determination of macromolecular complexes. It can be used to study atomic structure of 3-D crystals. The key requirement for a successful X-ray analysis is that the sample studied must grow to well-ordered 3-D crystals. However, due to this requirement, it is difficult to apply the technique of the X-ray crystallography in structural studies of viruses.Nuclear magnetic resonance (NMR) spectroscopy is a promising tool for studies of the atomic structure of small molecules and the macromolecule in solution. A unique feature of the technique is its ability to probe the dynamics of molecular conformations. However, the interpretation of the complex spectra obtained from the macromolecule with big molecular weight is exceptionally difficult. Therefore, NMR spectroscopy are normally used to study the structures of relatively small molecules with the molecular weight less than 35 Kda.Electron cryomicroscopy (cryoEM) is a fast emerging technique for three-dimensional structural reconstruction of macromolecular complexes. Through using this technique, images of frozen-hydrated macromolecules can be obtained by quickly freezing the specimen to the liquid nitrogen or liquid helium temperature, and then keeping the temperature below -150 C during the course ofimaging. CryoEM images are recorded using a low electron dose (<20e / A2/s) to minimize the radiation damage to the specimen. This allows investigation into three-dimensional structures of macromolecular complexes at a high resolution. In the conventional electron microscopy, the stabilization of the sample in microscope vacuum is achieved through negative stain, chemical fixation, and dehydration, etc. This results in the loss of the high-resolution structural information of the biological sample. Therefore, the conventional methods can only be used for studies at a very low-resolution level. In contrast, in cryoEM , the cooling of hydrated specimen is exceptionally fast, avoiding the formation of crystalline ice. It is just like that the whole sample is embedded in a vitreous ice with the depth of about 2um. The layer vitreous ice can not only prop up the sample as support membrane, but also preserve the water in frozen-hydrated sample in the high vacuum of the microscope, giving a better protection of the sample of hydrated macromolecular complexes. As a result, the sample can be placed in or near to in the physiological active state, and the high resolution structure information of the sample can be retained. This makes possible the 3D structure study of macromolecular complexes at a high resolution.Parvoviruses are able to infect human and mammalian cells, and to cause a lot of diseases. Many efforts have been made to study the biological characteristics of the parvoviruses. The biological characteristic of many vertebrate parvoviruses have been clarified, for example the genome organization, the structure of capsids protein of virus particle, and the relationship between the structure and its function. In particular, among these studies, the crystal structure of three vertebrate parvoviruses, canine parvovirus(CPV), feline panleukopenia virus(FPV), and minute virus of mice(MVM), have been determined at near-atomic resolution using the technique of X-ray crystallography. However, the studies of densoviruses are still limited. This thesis will contribute to this subject through investigation into the 3D structure of Periplaneta fuliginosa densovirus (pfDNV).Cockroaches, an ancient insect更多还原