摘要
利用指数富集配基的系统进化(SELEX)技术,以灭活的铜绿假单胞菌为靶标,从体外合成的96 nt随机ssDNA文库中筛选与铜绿假单胞菌特异结合的适体.在第12轮和第14轮与其它假单胞菌属进行反筛策略,并进行了适体的结合亲和力的测定,再分别利用ClustalX、Mega2和Mfold sever软件分析适体的一级和二级结构.研究结果表明,经过15轮筛选,随机ssDNA文库与铜绿假单胞菌结合的A值从0.022上升到0.448.反筛与未反筛的结合A比值最高比为53倍.经过第15轮反筛后的24个阳性克隆子测序,根据软件分析,其可分成10个家族.每个家族都有其共同的保守序列(除第10个家族),其中有2条序列几乎完全一致(F23和F47),同源性达到97%,A值分别高达1.598和1.508,Kd为14.55和77.46 nmol/L,间接说明适体与铜绿假单胞菌的结合力明显增高.
We have applied systematic evolution of ligands by exponential enrichment （SELEX） against the whole inactivate Pseudomonas aeruginosa （P. aeruginosa ）as a complex target to select single-stranded DNAligands （aptamers） from a random 96 nt library. The counter selection of ssDNA binding to other Pseudomonas was performed at 12 rounds and 14 rounds. After 15 rounds, the selected aptamers were cloned and sequenced, and the primary sequences and the structures were analyzed by ClustalX, Mega2 and Mfold sever softwares. The affinities of the cloned aptamers were also analyzed by enzyme-linked assays. The results showed that a significant enhancement of binding activities over background with the absorbance ranging from 0.022 to 0.448. The max ratio of absorbance units between the counter-selected and uncounter-selected was 53 folds. 24 of sequenced clones were classified into 10, and 9 of which were conserved sequences. Two out of the 24 sequences （ F23 and F47） sharing a 97 % identity were enriched. The absorbance were 1. 598 and 1. 508, and the equilibrium dissociation constant （Kd） were 14.55 and 77.46 nmol/L.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2009年第1期90-97,共8页
Chinese Journal of Biochemistry and Molecular Biology
基金
全军医学科学技术研究"十一五"计划资助项目(No.06G038)~~
关键词
指数富集配基的系统进化技术
铜绿假单胞菌
适体
systematic evolution of ligands by exponential enrichment （SELEX）
Pseudomonas aeruginosa
aptamer
