筛选结核分枝杆菌CFP-10抗原适体的研究  

Screening of aptamers to CFP-10 protein from Mycobacterium tuberculosis

作　　者：马占忠[1,2] 王玉炯[1] 秦莲花[2] 丁元生[2] 谢琴[1] 胡忠义[2] 

机构地区：[1]宁夏大学生命科学学院,宁夏银川750021 [2]同济大学附属肺科医院,上海市结核重点实验室,上海200433

出　　处：《中国病原生物学杂志》2008年第2期86-89,共4页Journal of Pathogen Biology

基　　金：上海市自然科学基金资助项目(No.06ZR14092);上海市科委重点专项资助项目(No.06DZ22034);同济大学科技发展基金(2005)资助项目

摘　　要：目的建立SELEX技术筛选结核分枝杆菌CFP-10抗原适体的方法,并获得CFP-10的高亲和性适体。方法体外构建长度为78个碱基的随机单链DNA(ssDNA)文库,以微孔板为筛选介质,采用SELEX技术筛选获得CFP-10的适体库。将适体库克隆、测序,用DNAMAN软件对其结构进行分析,利用酶联寡核苷酸吸附试验(enzyme-linkedoligonucleotide sorbent assay,ELOSA)测定亲和力。结果构建的ssDNA文库经过14轮筛选与CFP-10亲和力从0.273提高到1.265;克隆子测序,大多数长度与预期值相符。二级结构显示,口袋和茎环结构可能是适体与CFP-10结合的结构基础。结论成功建立了SELEX筛选技术,并初步获得了CFP-10的高亲和性适体。Objective To develop a SELEX screening method and obtain the high-afflnity aptamer to CFP-10 antigen from Mycobacterium tuberculosis. Methods An in vitro synthesized 78 mer random DNA library was subjected to selection by SELEX method against CFP-10 protein. The aptamers to CFP-10 protein were cloned and sequenced. DNA- MAN package was employed to analyze the conserved sequences and the second structure of the aptamers. Binding of the aptamers to the protein was examining by ELOSA. Results The binding assay between aptamers and the protein demonstrated that A values at 450nm were increased from 0. 273 to 1. 265 after 14 rounds selection. Most aptamers had the correct length after cloning and sequencing. Pocket and stem-loops was the basis of aptamers binding to CFP-10 protein by the analysis of structures. Conclusion The high-affinity aptamer binding to CFP-10 was successfully selected from the initial random ssDNA library.

关 键 词：结核分枝杆菌 CFP-10 SELEX 适体 

分 类 号：R378.911[医药卫生—病原生物学]