CN107179309B - 一种精氨酸激酶的检测方法 - Google Patents

一种精氨酸激酶的检测方法 Download PDF

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CN107179309B
CN107179309B CN201710362795.4A CN201710362795A CN107179309B CN 107179309 B CN107179309 B CN 107179309B CN 201710362795 A CN201710362795 A CN 201710362795A CN 107179309 B CN107179309 B CN 107179309B
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CN107179309A (zh
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赵玉芬
蔡华欢
黄碧玲
林荣灿
许鹏翔
刘艳
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Xiamen University
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    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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Abstract

一种精氨酸激酶的检测方法，涉及精氨酸激酶。提供由于激酶能够调节细胞内信号的传导、蛋白质的结构等，对于激酶的检测是非常重要的一种精氨酸激酶的检测方法。利用柠檬酸钠还原法合成胶体纳米金，再加入4‑MBA，然后通过离心除掉游离的4‑MBA，将沉淀重新分散在超纯水中；将底物肽溶于水中，再分别加入ATP和MgCl2，向底物肽中加入精氨酸激酶，得反应液后，置于水浴锅中恒温；将反应液加到纳米金中，混匀后进行拉曼光谱的测试，并最终得到标准曲线。

Description

一种精氨酸激酶的检测方法
技术领域
本发明涉及精氨酸激酶，尤其是涉及一种精氨酸激酶(McsB)的检测方法。
背景技术
由于磷氮键键能高，并且在酸性条件下容易水解，这使得人们对于精氨酸激酶的检测面临着巨大的挑战。自从2009年第一个精氨酸激酶被发现以来，许多工作都是致力于激酶的检测，目前主要的方法是免疫检测：利用磷酸化的精氨酸合成抗体，再利用抗体来检测精氨酸激酶(参见：Ouyang,H.,Fu,C.,Fu,S.Org Biomol Chem,2016,14,6，1925)。但是免疫的方法比较复杂，费时费力。因此需要一种操作步骤少、简单快速的激酶检测方法。
发明内容
本发明的目的在于提供由于激酶能够调节细胞内信号的传导、蛋白质的结构等，对于激酶的检测是非常重要的一种精氨酸激酶的检测方法。
本发明包括以下步骤：
1)利用柠檬酸钠还原法合成胶体纳米金，再加入4-MBA，然后通过离心除掉游离的4-MBA，将沉淀重新分散在超纯水中；
2)将底物肽溶于水中，再分别加入ATP和MgCl2，向底物肽中加入精氨酸激酶，得反应液后，置于水浴锅中恒温；
3)将步骤2)的反应液加到纳米金中，混匀后进行拉曼光谱的测试，并最终得到标准曲线。
在步骤1)中，所述合成胶体纳米金可合成尺寸为30nm的胶体纳米金；所述再加入4-MBA可过夜；所述4-MBA可选自对巯基苯甲酸等。
在步骤2)中，所述将底物肽溶于水中的质量浓度可为1mg/mL，底物肽可为2μL底物肽；所述再分别加入ATP和MgCl2可加入1μLATP和1μL MgCl2；所述反应液的体积可为100μL，所述置于水浴锅中恒温的温度可为40℃，水浴锅中恒温的时间可为3h。
在步骤3)中，所述反应液的浓度可为5μL，纳米金的体积浓度可为200μL。
本发明采用的分析化学基于表面增强拉曼光谱的方法，快速测定细胞裂解液中的精氨酸激酶含量的新技术。即以修饰了对巯基苯甲酸的纳米金为SERS基底，利用能够被精氨酸激酶所识别的底物肽来诱导纳米金聚集，当底物被磷酸化后，纳米金不能聚集，从而实现精氨酸激酶(McsB)的检测。
与现有方法相比，本发明具有以下突出优点：
1)提供一种快速检测精氨酸激酶的方法；
2)使用的药品都廉价易得；
3)操作简单，无需超低温或者超高温；绿色环保。
附图说明
图1为当加入不同浓度的精氨酸激酶McsB时，得到的表面增强拉曼光谱图。
具体实施方式
以下实施例将结合附图对本发明作进一步的说明。
本发明包括以下步骤：
1)利用柠檬酸钠还原法合成尺寸为30nm的胶体纳米金，再加入4-MBA过夜。然后通过离心除掉游离的4-MBA,最后将沉淀重新分散在超纯水中；
2)将底物肽溶于水中使得最终浓度为1mg/mL，取2uL此底物肽，再分别加入1uLATP和MgCl2，最后向底物肽中加入不同浓度的精氨酸激酶，最终体积为100uL,将100uL反应液置于40℃水浴锅中恒温3h；
3)取5uL上述反应液加到200uL纳米金中，充分混匀后进行拉曼光谱的测试，并最终得到标准曲线。
所述4-MBA选自对巯基苯甲酸。
图1给出当加入不同浓度的精氨酸激酶McsB时，得到的表面增强拉曼光谱图。

Claims (9)

1.一种精氨酸激酶的检测方法，其特征在于包括以下步骤：
1)利用柠檬酸钠还原法合成胶体纳米金，再加入4-MBA，然后通过离心除掉游离的4-MBA，将沉淀重新分散在超纯水中；
2)将底物肽溶于水中，再分别加入ATP和MgCl2，向底物肽中加入精氨酸激酶，得反应液后，置于水浴锅中恒温；
3)将步骤2)的反应液加到纳米金中，混匀后进行拉曼光谱的测试，并最终得到标准曲线。
2.如权利要求1所述一种精氨酸激酶的检测方法，其特征在于在步骤1)中，所述合成胶体纳米金是合成尺寸为30nm的胶体纳米金。
3.如权利要求1所述一种精氨酸激酶的检测方法，其特征在于在步骤1)中，所述再加入4-MBA过夜。
4.如权利要求1所述一种精氨酸激酶的检测方法，其特征在于在步骤2)中，所述将底物肽溶于水中的质量浓度为1mg/mL，底物肽为2μL底物肽。
5.如权利要求1所述一种精氨酸激酶的检测方法，其特征在于在步骤2)中，所述再分别加入ATP和MgCl2是加入1μLATP和1μL MgCl2。
6.如权利要求1所述一种精氨酸激酶的检测方法，其特征在于在步骤2)中，所述反应液的体积为100μL。
7.如权利要求1所述一种精氨酸激酶的检测方法，其特征在于在步骤2)中，所述置于水浴锅中恒温的温度为40℃，水浴锅中恒温的时间为3h。
8.如权利要求1所述一种精氨酸激酶的检测方法，其特征在于在步骤3)中，所述反应液的浓度为5μL。
9.如权利要求1所述一种精氨酸激酶的检测方法，其特征在于在步骤3)中，纳米金的体积浓度为200μL。
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