人凝血因子7a核酸适配体的筛选与鉴定
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摘要
人凝血因子7a (FVIIa)是外源性凝血途径的核心因子,检测参与凝血过程的FVIIa及针对FVIIa开发安全有效和经济的凝血剂和抗凝血剂是具有重要的意义。本研究基于指数富集配体系统进化技术(Systematic evolution of ligands by exponential enrichment,SELEX),采用酸纤维素膜法,通过11轮筛选成功分离出FVIIa的DNA适配体,并模拟了其二级结构。此适配体与FVIIa蛋白结合的表观解离常数为102 nmol/L。同时,基于所获得的高亲和力适配体,建立了基于适配体的荧光检测方法。本方法简单高效,对FVIIa蛋白的定量检测范围达到30～80 nmol/L,为人凝血因子7a的检测提供了一条新途径。非变性聚丙烯酰胺凝胶电泳和适配体的荧光检测方法结果表明,此适配体具有良好的选择性。
Human coagulation factor 7a(FVIIa) is the core factor for exogenous coagulation pathway. Developing safe, effective and economical coagulants or anticoagulants is still a challenge. In this study, a single-stranded DNA(ssDNA) aptamer for FVIIa was isolated based on systematic evolution of ligands by exponential enrichment(SELEX), which was based on the isolation of the sequences bound to the target protein, which would be absorbed on nitrocellulose membrane. The aptamer was isolated after 11 rounds of selection. The apparent dissociation constant of the aptamer to FVIIa was 102 nmol/L. The aptamer-based fluorescent assay for FVIIa was established, exhibing simplicity and efficiency. The quantitative detection range of FVIIa by this assay was 10-30 nmol/L, providing a new strategy for detection of FVIIa. The non-denaturation polyacrylamide gel electrophoresis and the aptamer-based fluorescent assay results both showed that the aptamer had good selectivity toward FVIIa.
Human coagulation factor 7a(FVIIa) is the core factor for exogenous coagulation pathway. Developing safe, effective and economical coagulants or anticoagulants is still a challenge. In this study, a single-stranded DNA(ssDNA) aptamer for FVIIa was isolated based on systematic evolution of ligands by exponential enrichment(SELEX), which was based on the isolation of the sequences bound to the target protein, which would be absorbed on nitrocellulose membrane. The aptamer was isolated after 11 rounds of selection. The apparent dissociation constant of the aptamer to FVIIa was 102 nmol/L. The aptamer-based fluorescent assay for FVIIa was established, exhibing simplicity and efficiency. The quantitative detection range of FVIIa by this assay was 10-30 nmol/L, providing a new strategy for detection of FVIIa. The non-denaturation polyacrylamide gel electrophoresis and the aptamer-based fluorescent assay results both showed that the aptamer had good selectivity toward FVIIa.
引文
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4 Nemerson Y.Blood,1988,71(1):1-8
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7 Hoffman M,Monroe D M,Oliver J A,Roberts H R.Blood,1995,86(5):1794-1801
8 Tuerk C,Gold L.Science,1990,249(4968):505-510
9 Ellington A D,Szostak J W.Nature,1990,346(6287):818-822
10 WANG Wei-Wei,LIU Su-Qin,XUE Yun,WANG Yan,YAN Chao.Chinese Journal of Chromatography,2017,35(1):99-104王薇薇,刘素琴,薛芸,王彦,阎超.色谱,2017,35(1):99-104
11 LIU Xiao-Hui,WANG Ze-Cheng,ZHANG Xiao-Bing,XU Dan-Ke.Chinese J.Anal.Chem.,2017,45(12):1971-1979刘晓辉,王则呈,张晓兵,许丹科.分析化学,2017,45(12):1971-1979
12 Liu J,Cao Z,Lu Y.Chem.Rev.,2009,109(5):1948-1998
13 Luo Y,Wang J,Yang L Y,Gao T,Pei R J.Sens.Actuators B,2018,276:128-135
14 DU Zhen-Ya,JIA Ru-Han,LI Wen-Hui,YANG Jing,XI Jing,SHI Da-Lin,HAN Yue-Wu.Chin.J.Biochem.Mol.Biol.,2018,34(10):1065-1072杜振亚,贾茹涵,李文慧,杨静,习静,石大林,韩跃武.生物化学与分子生物学报,2018,34(10):1065-1072
15 Wang J,Zhang Y J,Chen Y,Hong S N,Sun Y,Sun N,Pei R J.Talanta,2017,175:235-242
16 Liu D L,Zhang Z L,Yin Y J,Jia F,Wu Q P,Tian P,Wang D P.Talanta,2019,193:199-205
17 LI Hui,WANG Wei,DING Hong-Mei,DONG Jie,HUANG Ai-Xue,LUO Zhao-Feng,LI Jie,BAI Chen-Jun,LI Shao-Hua,SHAO Ning-Sheng.Lett.Biotech.,2018,29(4):459-464李慧,王玮,丁红梅,董洁,黄皑雪,罗昭锋,李洁,白琛俊,李少华,邵宁生.生物技术通讯,2018,29(4):459-464
18 Abdessamad T A,Laura F,Nick M,Jamal I,Pascale R,William J.Nucleic Acids Res.,2002,30:10e35
19 LIU Zhong-Cheng,ZHAO Li-Jun,ZHANG Yan-Fen,SHI Hai-Lang,XIE Yao.Acta Pharm.Sin.,2012,47(12):1605-1611刘中成,赵丽君,张艳芬,时海浪,解瑶.药学学报,2012,47(12):1605-1611
20 WANG Kun,TAO Zhan-Hui,XU Lei,LIU Ya-Qing.Chinese J.Anal.Chem.,2014,42(2):298-304王昆,陶占辉,徐蕾,刘亚青.分析化学,2014,42(2):298-304
21 DU Yu-Lin,MO Liu-Ting,YI Ya-Sha,QIU Li-Ping,TAN Wei-Hong.Chinese J.Anal.Chem.,2017,45(12):1757-1765堵玉林,莫柳婷,易娅莎,邱丽萍,谭蔚泓.分析化学,2017,45(12):1757-1765
22 Stojanovic M N,de Prada P,Landry D W.J.Am.Chem.Soc.,2001,123(21):4928-4931
23 Nutiu R,Li Y.J.Am.Chem.Soc.,2003,125(16):4771-4778
24 Song W J,Strack R L,Sevensen N,Jaffrey S R.J.Am.Chem.Soc.,2014,136(4):1198-1201
25 Rusconi C P,Yeh A,Lyerly H K,Lawson J H,Sullenger B A.Thromb.Haemost.,2000,84:841-848