金葡菌特异性配基的筛选及初步应用

发布时间：2018-01-06 09:35

本文关键词：金葡菌特异性配基的筛选及初步应用　出处：《中国人民解放军军事医学科学院》2008年硕士论文　论文类型：学位论文

【摘要】： 指数式富集的配体系统进化(SELEX)技术是近年来发展起来的研究核酸结构、功能及进化的一种新的组合化学技术。它在基础医学和临床诊断方面具有广泛的应用前景。本研究的目的是建立一种以完整细菌为靶子的消减SELEX技术,并筛选出与金黄色葡萄球菌特异结合的一组ssDNA配基(aptamer),为致病微生物的快速检测提供理论基础及实验依据。我们首先设计了带有茎环的下游引物,通过获得不同长度单链产物的PCR技术原理,建立了一种新的ssDNA制备方法。然后采用随机区为45个碱基、全长88nt的ssDNA文库,以金葡菌8325-4菌株为目的靶,链球菌A5005菌株和表皮葡萄球菌26069菌株为消减靶,进行了七轮的SELEX筛选。对每一轮文库标记同位素,结合实验证实第五轮文库富集程度最高。将富集的第5轮文库进行克隆测序,分别采用MEME在线软件和RNA STRUCTURE软件对序列的结构进行分析,得到11条稳定序列。随后利用流式细胞仪和激光共聚焦显微镜找出了5条特异性强的aptamer,其中结合效果最好的两个aptamer的解离常数均在nM水平。在以上工作的基础上我们对组合aptamer的性能进行研究,结果显示出,与单一的aptamer相比,组合aptamer能够提高对金葡菌的敏感性和检出率。本研究为建立一种新的致病微生物快速检测手段奠定了基础。
[Abstract]:Systematic evolution of ligands by exponential enrichment (SELEX) technology is the study of nucleic acid structure developed in recent years, a new combinatorial chemistry function and evolution. It has wide application prospect in basic medicine and clinical diagnosis. The purpose of this study is to establish a complete SELEX target to reduce bacteria, and selected a group of ssDNA ligand binding with Staphylococcus aureus specific (aptamer), and provide a theoretical basis and experimental basis for the rapid detection of pathogenic microorganisms.
We designed primers with stem loop, the principle of PCR technology to obtain different length single product, establish a new preparation method of ssDNA. Then the random region is 45 bases, ssDNA Library of full-length 88nt, Staphylococcus aureus 8325-4 strain as target, chain coccus aureus strain A5005 and epidermal 26069 strains of Staphylococcus aureus were screened for abatement target, the seven round of the SELEX. For each round of isotope labeling experiments confirmed that the library, combined with the fifth round of the library enriched the highest degree. The fifth round library enrichment were cloned and sequenced, respectively using MEME online software structure and RNA STRUCTURE software for sequence analysis, get 11 stable sequence. Then using flow cytometry and confocal microscopy to identify 5 specificity aptamer, the dissociation constant of binding effect of two aptamer were the best at the level of nM in the above work. Based on our research on the performance of combined aptamer, the results showed that compared with a single aptamer, combined aptamer could improve the sensitivity and detection rate of Staphylococcus aureus. This study laid the foundation for establishing a new rapid detection method for pathogenic microorganisms.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R378

【引证文献】