【作者】 张晖；

【导师】 田杰； 张静；

【作者基本信息】 重庆医科大学 ， 儿科学， 2008， 硕士

【摘要】 背景轮状病毒(rotavirus,RV)是导致全世界婴幼儿严重腹泻病的常见病原之一,每年可导致大约50万患儿死亡。无论发达国家还是发展中国家,RV的感染率和发病率都很高。因此迫切需要开发有效的疫苗和治疗策略来对抗病毒。而这些研究的基础又需要从根本上认识病毒作用于宿主细胞的分子机制。RV感染细胞的第一个关键步骤是病毒与被感染细胞表面的病毒受体结合。由于RV受体在宿主细胞上数量较少、性质复杂以及技术方法的局限性,其性质迄今不清。全长cDNA文库构建、测序和功能注释是了解生物体结构和功能的一种重要方法之一,它有助于鉴定其基因组序列中的外显子-内含子的边界,分析基因编码区和理解基因在转录和翻译水平的功能,是分析人类基因表达图谱、功能和结构的重要资源。文库筛选作为一种获取未知功能基因的手段,对于研究病毒作用于人体的分子机制起着非常重要的作用。酵母双杂交技术自创立以来已成为研究蛋白质之间相互作用的重要方法之一。传统的酵母双杂交方法所研究的蛋白质间相互作用是在细胞核内完成的,而近年来在此基础之上发展起来的酵母双杂交衍生系统---Ras募集系统(RRS),能够将蛋白质的相互作用定位于细胞膜上,更具备了灵敏度高、假阳性少等突出优点。RRS的应用原理为:酵母温度敏感株由于缺乏鸟苷酸交换因子(GEF)cdc25-2,而不能将外来信号传递给膜上的Ras蛋白,致使该种突变株在36℃不能存活;如果人为引入正常的GEF(Ras蛋白),并使得GEF能与膜足够接近,则可弥补这一缺陷,激活Ras信号途径,使酵母细胞能够在36℃生长。目的在本研究中,我们构建一个婴幼儿小肠组织的全长cDNA文库并将其插入RRS系统中的pUra-MΔpolyA载体,构建成猎物载体,将其与本课题组前期构建好的RV外壳蛋白VP4诱饵载体pMet425-Myc-Ras-VP4共转化酵母温度敏感菌株cdc25-2,通过RRS筛选文库中与RVVP4相互作用的蛋白质分子,探寻轮状病毒与肠道宿主细胞相互作用的分子机制。方法1.从婴幼儿小肠组织中提取总RNA,并纯化mRNA,反转录合成第一、二链,连接EcoRI人工接头,分级分离后,除去小于500bpcDNA片段,与pUra-MΔpolyA连接后,CaCl2法转化感受态细胞DH5α,测定文库大小并EcoRI/XhoI双酶切鉴定插入cDNA片段的大小。2.将诱饵载体pMet425-Myc-Ras-VP4与猎物载体pUra-MΔpolyA-cDNA共转化酵母温度敏感菌株cdc25-2,通过改变培养基的营养成分及生长温度,去除可能的假阳性结果,筛选出与RVVP4相互作用的蛋白质分子。结果1.成功构建含有6×107重组子的婴幼儿小肠组织全长cDNA文库,插入片段的大小范围在0.5-2 kb之间,平均长度1.5kb。2.通过Ras募集系统,筛选出阳性克隆,序列测定尚在进行中。结论1.首次构建了一个婴幼儿小肠组织全长cDNA文库,并验证了插入片段大小符合进一步筛选所需,库容量亦足够大,是一个高质量的文库。2.利用RRS筛选系统筛选出了婴幼儿小肠组织全长cDNA文库中可能与RV外壳蛋白VP4相互作用的受体蛋白。更多还原

【Abstract】 Background Rotavirus (RV) is the main pathogen of infant’s diarrhea, accounting for as many as five million deaths annually. Because the morbility and infection rate are high either in developed or in developing country, it is more necessary to develop an effective vaccine and therapeutic strategies to combat virus. But the premise is we need a fundamental understanding of the molecular mechanism between virus and the host cells.The first and necessary step for RV infection is its binding to the receptors on the cell membrane. Up to now, the natures of those receptors are unclear because few quantities on the host cell, complex natures as well as technique’s limitation. Full-length cDNA is not only helpful in appraising the extron - intron boundary in its genome team sequence, analyzing gene code area and understanding gene’s copying and translation function, but also can be used to analyze the human genome expression profile, function and structure.The yeast two-hybrid system has been one of the important methods for research of interaction between proteins since it was founded. Traditional two-hybrid locates the interaction in the nucleus, while the Ras Recruitment System (RRS) developed in recent years can locate which on the cell membrane and has more advantages such as high sensitivity and less false positive. The principle of the RRS is as follow: In the yeast temperature-sensitive cdc25-2 strain, the yeast Ras is rendered inactive at the restrictive temperature (36℃) due to lack of a functional cdc25-2 guanyl nucleotide exchange factor. But if we introduce a normal GEF (Ras protein) which could be localized on membrane through protein-protein interaction, the yeast temperature sensitive mutant strain cdc25-2 can grow at 36℃because of Ras signaling pathway activation.Objectives In our study, we constructed a full-length cDNA library of infant’s small intestine tissue and then cloned it to the plasmid pUra-MΔpolyA to obtain a prey plasmid suitable for RRS. To understand the molecular mechanism of interactions between RV and host cell, we cotransformed bait plasmid (pMet425-Myc-Ras-VP4) and prey plasmid (pUra-MΔpolyA-cDNA) into the yeast temperature sensitive mutant strain cdc25-2 to screen the proteins interacted with RV VP4 by RRS.Methods1. Total RNA from small intestine tissue was extracted by Trizol. After mRNA purified, cDNA was synthesized by reverse transcription, and cDNA fragments larger than 500bp were ligated with dephosphorylated arms of pUra-MΔpolyA vectors. The recombination vectors were transformed into DH5αby CaCl2. Calculating the library capacity and identifing the size of insert fragment by digesting with restriction enzymes EcoRI/XhoI.2. Bait plasmid (pMet425-Myc-Ras-VP4) and prey plasmid (pUra-MΔpolyA-cDNA) were cotransformed into the yeast temperature sensitive mutant strain cdc25-2. Screening the proteins interacted with RV VP4 by changing the components of the midum and environmental temperature to get rid of potential false positive.Results1. The full-length cDNA library contained 6×107 recombine clones. The insert size ranged from 0.5 to 2 kb, average 1.5kb.2. Screening positive clones by RRS, and detecting the sequences.Conclusions1. A high quality full-length cDNA library from infant’s small intestine tissue was first constructed successfully which can be used for screening by RRS.2. The possible protein receptor from cDNA library of infant’s small intestine tissue were found out which interacted with RV coat protein VP4 by RRS, waiting for the results of sequencing.更多还原