丙型肝炎病毒内部核糖体进入位点的三级结构研究

【作者】 宁俊红；

【导师】 Alastair Murchie；

【作者基本信息】 复旦大学 ， 药物化学， 2008， 硕士

【摘要】 丙型肝炎病毒(hepatitis C virus)的基因组是大约9.5kb的单链RNA,它能编码一个含有3000个氨基酸的蛋白质;它的基因组很复杂,现在已经知道有6种基因型和100种亚基因型,这些基因型的同源性少于65%,但这些基因型的5’—非翻译区域的同源性大于85%,其中内部核糖体进入位点(Internal RibosomeEntry Site IRES)的序列几乎完全保守。IRES坐落于mRNA的5’—非翻译区域(5’—UTR),它能启动不依赖帽式结构病毒翻译的开始。现在人们已经知道了IRES的一级结构和二级结构,并且知道它的一级结构和二级结构对HCV翻译起始很重要。虽然还不完全知道它的三级结构以及相关功能,但是已经知道它的三级结构形成与离子浓度、温度和RNA的浓度有关。IRES包括390个碱基,由一系列的连接点(junctions)、环(100ps)和茎(stems)组成,可大致分为4个结构域和2个相互独立的结构群。IRES的三级结构也许就是由这些结构域相互作用形成的。目前人们已经用冷冻电镜法、结晶法、X—衍射法和核磁共振等方法对IRES的三级结构做了一些研究,但这些方法都有各自的局限性,而且有时产生了不一致甚至相反的结果,这很可能是IRES在形成三级结构的过程中它的空间构象是不断变化的。本课题首先设计实验中所用的HCV IRES的有关序列、合成并纯化设计的DNA序列、应用体外转录法得到RNA序列并纯化此RNA序列、去磷酸化所有RNA序列并标记部分RNA序列,然后研究在不同的离子浓度、不同的RNA浓度以及不同的温度条件下,IRES的结构域Ⅲ(domainⅢ)的连接点(iunction)在不同的位置对结构域Ⅲ形成的影响、不同大小的结构域Ⅱ与完整的结构域Ⅲ和不完整的结构域Ⅲ的相互作用以及结构域Ⅲ的连接点中有点突变时对结构域Ⅲ形成的影响。更多还原

【Abstract】 Hepatitis C Virus is a single RNA who has about 9.5kb genome and can encode one protein containing 3000 amino acids;Its genome is very complicate,Now we have known there has six genetypes and 100 sub-genetypes,the homology of these genetypes are less 65%,but the homology of 5’—untranslated region of these genetypes are more 85%,moreover the sequences of Internal Ribosome Entry Site are conservative wholly.IRES locates in the mRNA 5’—untranslated region who can promote translation initiation of the viral message via a 5’—cap—independent mechanism.Nowaday,we know the primary and secondary structure of IRES that is very important to the translation of HCV.Although we don’t know its tertiary structure and relative function totally,we have known the tertiary structure is concerned with ionic concentration、RNA concentration and temperature.IRES is consist of a serial of junctions,loops and stems and contains 390kb,It can be divided four domains and two interdependent frameworks.The interaction of these domains may form the tertiary structure of IRES.There have some research about the tertiary structure of IRES through electron microscopy,crystal,X-ray and NMR etc,but the results obtained from those methods are discrepancy and contradictory,It may be the reason that the space conformation of IRES tertiary structure is changing.In my experiment we design relevant sequences of HCV IRES,make and purify DNA sequences,make RNA sequences with in vitro transcription and purify them, dephosphorylate all RNA sequences and label some RNA sequences firstly.Then under difference ionic concentration,difference RNA concentration and difference temperature,we will study the influence of junction on the domainⅢand the interaction between difference size domainⅡand intact domainⅢand incomplete domain Ⅲ;Finally we will study the influence of mutation within junctions on the domainⅢ.更多还原