改良SELEX技术筛选福氏志贺菌2a特异性适配体  

Characteristics and screening of aptamers to Shigella flexneri 2a based on modified whole bacteria reduction SELEX

作　　者：王国臻 王玉格 兰春阳 李翔 李明成 付桂莲 WANG Guo-Zhen;WANG Yu-Ge;LAN Chun-Yang;LI Xiang;LI Ming-Cheng;FU Gui-Lian(School of Medical Techonology,Beihua University,Jilin 132013,China)

机构地区：[1]北华大学医学技术学院,吉林132013

出　　处：《中国免疫学杂志》2021年第10期1226-1230,共5页Chinese Journal of Immunology

基　　金：国家自然科学基金项目(81541141);北华大学研究生创新计划(北华研创合字[2018]036)。

摘　　要：目的:构建与福氏志贺菌2a高亲和性、特异性结合的DNA寡核苷酸适配体库。方法:体外合成76 nt高通量随机序列的单链DNA寡核苷酸文库(ssDNA),运用全细菌指数富集的配体系统进化技术(whole-bacteria SELEX),福氏志贺菌2a作为靶标与文库ssDNA反应;通过优化PCR反应,将Lambda酶外切及葡聚糖凝胶层析纯化技术用于次轮反应文库的制备,荧光分光光度计跟踪筛选过程,流式细胞仪克隆测序特异性单链核酸适配体,生物信息学技术分析其一、二级结构。结果:经过PCR技术10轮正向筛选和6轮消减菌筛选,通过Lambda酶外切及葡聚糖凝胶层析纯化,ssDNA平均回收率高达72.33%。依据一级结构的同源序列建立了36个寡核苷酸探针组群。流式细胞仪分析获得1条与福氏志贺菌2a特异性结合的适配体(命名ZH-21)。结论:基于全菌消减SELEX技术,结合Lambda酶外切及凝胶层析纯化技术,建立一种快速、稳定、高效、特异性高和成本低的筛选福氏志贺菌2a特异性单链核酸适配体的方法。Objective:A single strand aptamer which can recognize Shigella flexneri 2 a quickly, stably and specifically was screened and identified.Methods:In vitro synthesis of 76 nt high-throughput random sequence single-stranded DNA oligonucleotide library(ssDNA), the strain of Shigella flexneri 2 a was taken as the target bacteria, and several kinds of bacteria with similar pathogenicity were taken as the reducing bacteria using the whole bacteria reduction SELEX technology.The modified single strand aptamers of Shigella flexneri in vitro were rapidly screened by improved gel chromatography. The primary and secondary structures of aptamers were analyzed by fluorescence spectrophotometer, flow cytometer, clone sequencing, and clinical application of aptamers was identified.Results:After ten rounds of whole bacterial subtractive SELEX screening,the results of fluorescence spectrophotometer showed that the enrichment process was saturated,and dextran gel(Sephacryl)chromatography was added to the screening process,which greatly shortened the preparation time. By sequencing,flow cytometry and fluorescence microscopy,a specific binding aptamer to Shigella flexneri was obtained.Conclusion:A 2 a specific single strand aptamer of Shigella flexneri was screened based on gel chromatography purification technology,and a rapid,stable,high efficiency,high specificity and low price detection method for Shigella flexneri 2 a was established.

关 键 词：全菌消减SELEX技术 福氏志贺菌2a 凝胶层析纯化技术 适配体 

分 类 号：R378[医药卫生—病原生物学]