【作者】 李华；

【导师】 邓乐；

【作者基本信息】 湖南师范大学 ， 微生物学， 2011， 硕士

【摘要】 产肠毒素大肠杆菌(ETEC) K88所引起的仔猪腹泻是一种十分常见的疾病,给仔猪和人类的健康带来极大的威胁,并且能造成十分严重的世界范围的经济损失。菌体表面的黏附性菌毛是ETEC K88致病的主要原因,ETEC K88通过菌毛吸附宿主肠道黏膜上皮细胞的相应受体,定居在该吸附区域,进而进行繁殖并产生毒素,损伤宿主小肠黏膜,引起宿主吸收分泌功能失常,导致腹泻。适配体是一段核酸序列,通过指数富集配基的系统进化技术(SELEX),从体外人工合成的ssDNA或RNA随机序列库中,循环筛选获得,多种多样的空间构象是其与靶分子相互结合的基础。适配体具有价格低廉,易合成,分子小,多样性广等特点,能特异性地结合细胞内与细胞外的靶分子物质,广泛的应用于基础研究,临床诊断治疗,药物研发和分子检测等领域。本论文主要包括以下内容：通过体外SELEX技术,以ETEC K88菌毛蛋白作为靶分子,采用BSA作为反筛选靶分子进行反筛,从库容量为460的随机ssDNA文库当中筛选获得了一组能够高特异性、高亲和性结合ETEC K88菌毛蛋白的适配体,这是国内外ETEC K88菌毛蛋白适配体的首例报道。利用软件对筛选得到的适配体进行一级结构同源性和二级结构相似性分析,将适配体划分为四个家族。随机从四个家族中挑选四条序列分析其与ETEC K88菌毛蛋白的亲和力,由于荧光具有高灵敏性,我们采用荧光仪作为检测仪器。四条适配体中具有最高亲和力的一条被用来进一步分析其与ETEC K88的结合能力。由于只有ETEC K88能够表达ETEC K88菌毛蛋白,ETEC K88菌毛蛋白的适配体成功的实现对ETEC K88的定性和定量检测。这一检测方法不仅能够用于检测ETEC K88,也为其他细菌的快速、实时检测提供了一种新的思路。更多还原

【Abstract】 It has been reported that the enterotoxigenic Escherichia coli (ETEC) K88 strains may cause diarrheal disease in farming animals. It is a huge threat for the health of human beings and animals. Fimbriae play an essential role in the pathogenesis of ETEC K88 infection by mediating adhesion to epithelial cells. ETEC K88 adhere to host’s cell by fimbriae recognizing their receptor, and localize in local tissue and produce toxin or destroy tissue, leading to diarrhea.Aptamers are oligonucleotides (DNA or RNA) which obtained by SELEX (Systematic evolution of ligands by exponential enrichment). SELEX was on the basis of the fact that some unique RNA/DNA molecules can bind to the target molecules with high affinity and specificity. It is applied to identify these unique RNA/DNA molecules from very large population of random sequence oligomers. The interaction between oligonucleotides and the target molecules is based on the diversity in three-dimensional structures of oligonucleotides in the library. With their low cost, easy and quick preparation, small size and versatility, aptamers are ideal tools for the assay of intracellular and extra cellular targets. Aptamers have been widely used in the fields of basic research, clinical diagnosis, drug development and the detection of molecules.This paper includes the following researchs: The first group of ssDNA aptamers that are highly specific to ETEC K88 fimbriae protein was obtained from an enriched oligonucleotide pool by SELEX procedure, during which K88 fimbriae protein was used as the target and BSA as counter targets. They were then grouped under different families based on the similarity of their secondary structure and the homology of their primary sequence. Four sequences from different families were deliberately chosen for further characterization by fluorescence analysis. With the advantage of high sensitivity, fluorescence photometer was selected as ssDNA quantification method during the SELEX process. Aptamers with the highest specificity and affinity were analyzed to evaluate binding ability with ETEC K88. Since ETEC K88 is the only type of bacterium which expressed abundant K88 fimbriae, the selected aptamers against ETEC K88 fimbriae protein were able to specifically identify ETEC K88 among other bacteria. The method of detecting ETEC K88 by aptamers can also be applied to bacteria other than ETEC K88.更多还原