肺表面活性蛋白A适配体的筛选与鉴定  

Screening and Identification of Aptamers Aganinst Pulmonary Surfactant Protein A

作　　者：刘利娟[1] 陈怡[1] 王伟[1] 陈超[1] 高明昊[1] 张晓青[1] 张娟琨[1] 

机构地区：[1]天津科技大学生物工程学院,教育部工业微生物重点实验室,天津市工业微生物重点实验室,天津300457

出　　处：《分析化学》2013年第11期1659-1663,共5页Chinese Journal of Analytical Chemistry

基　　金：天津市科委项目(No.12ZXCXSY07500);天津市滨海新区科委项目(No.2012-XJR21047);科技部项目(No.12C26211600515)资助

摘　　要：利用指数式富集的配基系统进化(Systematic evolution of ligands by exponential enrichment,SELEX)技术筛选肺表面活性蛋白A(Pulmonary Surfactant Protein A,SP-A)的寡核苷酸适配体。通过对循环次数、退火温度及引物比例的优化,建立了适合的筛选体系;筛选产物经克隆测序后,用DNAMAN软件初步分析其结构。最终确定以12个循环,55℃为退火温度及80∶1为引物比的反应条件;经过9轮循环筛选,随机ssDNA库与SP-A的结合率从1.3%上升到33%;AP-3和AP-2适配体的随机区含有保守序列TAC-GT,AP-3和AP-1适配体的随机区含有保守序列ACAG,且各适配体的二级结构均以茎-环结构为主。Aptamers against surfactant protein A（SP-A） were screened by evolution of ligands by exponential enrichment（SELEX）. Through the optimization of critical PCR and asymmetric PCR parameters including cycle numbers, annealing temperature and ratio of primers, a suitable screening system was established. The optimum conditions were as follows. The optimal cycle was 12, the optimal annealing temperature was 55 ~C, and the optimal ratio of primer was 80： 1. The structure of aptamers was analyzed by DNAMAN software after being cloned and sequenced. After 9 rounds selection, the binding rate of ssDNA to SP-A increased from 1.3% to 33%. The aptamer AP-3 and AP-2 had the same sequence TAC-GT that was called conserved sequence; the aptamer AP-3 and AP-1 had the same sequence ACAG. And all of the aptamers＇ three- dimensional shape characterized by stems and loops.

关 键 词：肺表面活性蛋白A 指数式富集的配基系统进化 寡核苷酸适配子 

分 类 号：R341[医药卫生—基础医学]