摘要
以凝血酶适体(aptamer)为例,利用适体和核酸外切酶特性,通过定量PCR扩增建立一种高灵敏的蛋白质检测方法.首先合成3段寡核苷酸序列即凝血酶适体探针,上游连接子和下游连接子.将适体探针与凝血酶温育结合后,再加入核酸外切酶I降解未能结合的探针.接着将保护下来的探针与连接子杂交、连接和对连接产物进行定量PCR .分别建立连接产物标准品浓度与Ct 值的标准曲线和凝血酶浓度与连接产物浓度的标准曲线,通过定量PCR对凝血酶进行定量.结果显示,基于适体的外切酶保护凝血酶检测方法灵敏度较高,连接产物标准品浓度的对数值和Ct 值之间的方程为y =- 2 95x + 33 6 5 (R2 =0. 990 ,P <0 .0 1) ;凝血酶浓度和连接产物浓度对数值之间的方程为y =0 94x - 0 . 2 9(R2 =0 . 998,P <0 . 0 1) ,还对可能影响检测的有关参数举行了探讨.
Aptamers are single-stranded oligonucleotides with a length of tens of nucleotides and single stranded DNA aptamers have a very highly ordered tertiary structure,which allows them to form stable and specific complex with different targets such as protein,nucleic acid.In this study,we used one of thrombin-binding DNA aptamers to develop a high-sensitive new method to detect proteins based on realtime-PCR and the nature of aptamers and exonuclease I. Three oligonucleotide sequences (probe of thrombin aptamer and two connectors) were synthesized first. When thrombin and its aptamer incubated together, the thrombin bound an aptamer and thereby was protected from degraded by exonuclease I. Subsequently, the protected aptamer probes acted as linkers to join two free connectors, which contained sequences matching the probe. The joined products could reflect the identity and amount of the target protein. Two regression equations of series of ligation product standard and thrombin were built after realtime-PCR and we can quantify the thrombin of samples via them. we found a very high sensitivity in this method and a linear relation between threshold cycles and corresponding logarithms of different standard concentration(y=-2.95x+33.65, R2 =0.990) and between logarithms of ligation product and the corresponding thrombin(y=0.94x-0.29, R2 = 0.998). Furthermore, some optimal reactive conditions including optimal reactive time,of exonuclease 1 and T-4 DNA~ ligase and optimal ratio of aptamer to connector were also defined,respectively.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2005年第3期421-426,共6页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金资助项目(No.30170051
No.30170903)~~
关键词
蛋白质检测
适体
酶切保护分析
实时PCR
凝血酶
protein detection, aptamer, exonuclease protection assay, realtime-PCR, thrombin
