摘要
基于配体指数富集系统进化技术筛选闭锁连接蛋白-1的核酸适配体,最终筛选到15条ZO-1的核酸适配体(APTZO-1).排除线性结构与相似结构后,采用酶联免疫吸附法表征了9条APTZO-1与ZO-1亲和力的大小,排序依次为:APTZO-18>APTZO-12>APTZO-17>APTZO-113>APTZO-11>APTZO-15>APTZO-115>APTZO-112>APTZO-14.采用Cy5荧光基团标记APTZO-18(Cy5-APTZO-18),证实了其能对人结直肠腺癌细胞(Caco-2)与人脑微管内皮细胞(hCMEC/D3)的ZO-1进行荧光定位与半定量分析,且与基于ZO-1抗体法的免疫荧光与蛋白印迹实验结果基本相似.本研究首次筛选到了ZO-1的系列核酸适配体,有望为ZO-1的荧光标记与半定量分析提供一种新型的、特异性高的工具分子.
To select the Aptamer(APT)of Zonula Occludens-1(ZO-1),a systematic evolution technique of lig-ands was used based on exponential enrichment(SELEX),and 15 APTZO-1 were selected as Aptamers finally.Ex-cluding linear and similar structures,an enzyme-linked immune-sorbent assay was used to evaluate the affinity between 9 APTsZO-1 and ZO-1.The analysis results show the order of affinity,APTZO-18>APTZO-12>APTZO-17>APTZO-113>APTZO-11>APTZO-15>APTZO-115>APTZO-112>APTZO-14,signed with Cy5 fluorescent labeling,for instance,Cy5-APTZO-18 labeling for APTZO-18.The enzyme-linked immune-sorbent method can be used for fluorescence localization and semi-quantitative analysis of ZO-1 for human epithelial colorectal adenocarcinoma cells(Caco-2)and human brain capillary endothelial cells(hCMEC/D3).And the result is identical with results of immunofluorescence and Western blotting based on ZO-1 antibody methods.In the study,a series of APTs for ZO-1 are firstly screened and expected to provide a new tool molecule with high specificity for fluorescent la-beling and semi-quantitative analysis of ZO-1.
作者
李玉娟
张瀚文
刘珊
王佳平
李勇枝
LI Yujuan;ZHANG Hanwen;LIU Shan;WANG Jiaping;LI Yongzhi(School of Life Science,Beijing Institute of Technology,Beijing 100081,China;Key Laboratory of Astronaut Health Center,Astronaut Center of China,Beijing 100094,China)
出处
《北京理工大学学报》
EI
CAS
CSCD
北大核心
2023年第8期870-877,共8页
Transactions of Beijing Institute of Technology
基金
国家自然科学基资助项目(81973572)
航天员健康中心重点实验室基金项目(AHCC2021KF004)。
关键词
闭锁连接蛋白-1
配体指数富集系统进化
核酸适配体
荧光标记
半定量分析
Zonula Occludens-1(ZO-1)
systematic evolution of ligands by exponential enrichment(SELEX)
Aptamer(APT)
fluorescent labeling
semi-quantitative analysis
