一种核酸适配体高敏检测方法的建立及应用评价  

Establishment and application of a high sensitivity detection method for nucleic acid aptamer

作　　者：陈连娣 朱雯婷 李振华 袁中文 陈亦清 林彩燕[1] 程媛媛 严鹏科[1] CHEN Liandi;ZHU Wenting;LI Zhenhua;YUAN Zhongwen;CHENYiqing;LIN Caiyan;CHENG Yuanyuan;YAN Pengke(Pharmacy Department,The Third Affiliated Hospital of Guangzhou Medical University,Guangzhou 510150,Guangdong,China;Joint Laboratory for Translational Cancer Research of Chinese Medicine,Guangzhou University of Chinese Medicine,Guangzhou 510006,Guangdong,China)

机构地区：[1]广州医科大学附属第三医院药剂科,广东广州510150 [2]广州中医药大学国际中医药转化医学研究所,广东广州510006

出　　处：《中南医学科学杂志》2019年第2期198-201,共4页Medical Science Journal of Central South China

基　　金：广东省科技重大项目(No.2015B02023315);广州市科技重大项目(No.201604020166)

摘　　要：本文建立一种基于免疫印迹的核酸适配体高敏定量分析的检测方法,并探讨其在适配体药物靶向性研究中的应用价值。首先用聚丙烯酰胺凝胶电泳进行核酸适配体的分离,然后利用电转的方法将凝胶上的核酸适配体转移至PVDF膜上,先后孵育Rabbit Anti-Biotin和HRP-Streptavidin抗体,最后利用化学发光仪进行成像。结果显示核酸适配体可以通过该方法进行定量检测分析,且该方法检测灵敏度高,能够识别荧光染料法检测不到的浓度范围。此外,该方法只识别被生物素修饰的核酸适配体,特异性强。结果提示,核酸免疫印记法可以作为核酸适配体药物靶向性研究的一个定量检测手段。To establish a high sensitivity quantitative detection method for nucleic acid aptamer based on western immunoblotting, and assess its application in targeted detection of aptamer drugs. Aptamer was separated by polyacrylamide gel electrophoresis. Then, the aptamer on the polyacrylamide gel was transferred to PVDF membranes, followed by incubating with Rabbit Anti-Biotin and HRP- Streptavidin. Finally, the blot signals were detected by a Bio-Rad Ultraviolet Imaging System. Results show that aptamer could be detected by nucleic acid immunoblotting. And this method was high sensitive for it can detect the low concentration range which cannot be detected by fluorescence dye. In addition, there is a high specificity as it only recognized the aptamer modified by biotin. Nucleic acid immunoblotting can be contributed to quantify the targeting of aptamer drugs.

关 键 词：核酸适配体 免疫印记 荧光染色 生物素-亲和素系统 

分 类 号：R-331[医药卫生]