CN110257388B - 一种特异性识别精氨酸的多肽适配体及应用 - Google Patents

一种特异性识别精氨酸的多肽适配体及应用 Download PDF

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CN110257388B
CN110257388B CN201910604881.0A CN201910604881A CN110257388B CN 110257388 B CN110257388 B CN 110257388B CN 201910604881 A CN201910604881 A CN 201910604881A CN 110257388 B CN110257388 B CN 110257388B
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polypeptide
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冯泽猛
贺玉敏
邓磊
曹忠
肖忠良
印遇龙
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Abstract

本发明公开了一种特异性识别精氨酸的多肽适配体及应用，所述多肽适配体的序列如SEQ ID NO.1所示。本发明通过基于计算机辅助的分子对接模拟预测，筛选得到可以与L‑精氨酸特异性结合的多肽适配体，并通过等温滴定量热仪进行验证。该多肽适配体具有稳定性好、结合能力强、特异性强、生产成本低的优点。

Description

一种特异性识别精氨酸的多肽适配体及应用
技术领域
本发明涉及属于生物物理学、生物化学与分子生物学以及分析化学的交叉领域，具体涉及一种特异性识别精氨酸的多肽适配体及应用。
背景技术
L-精氨酸是组成蛋白质的基本组成成分之一，在动物体内参与鸟氨酸循环，促进尿素的形成，使人体内产生的氨经鸟氨酸循环转变成无毒的尿素，经尿排出，从而降低血氨浓度。此外，L-精氨酸对维持体内蛋白质的电性具有重要贡献，和赖氨酸共同为碱性氨基酸。近年来有研究表明，L-精氨酸可以被当做是重要的疾病相关的生物标志物，由于肝细胞癌患者体内的琥珀酸合酶无法被合成，因此常表现出体内L-精氨酸浓度较低。此外，L-精氨酸也是动植物组织中营养状态的指标，当营养状态异常时，精氨酸含量也表现出不同水平的差异。与其他氨基酸一样，氨基酸是食品中营养价值水平高低的重要衡量指标。目前，精氨酸的分析方法主要有两类：其一是利用大型仪器，比如液相色谱仪、氨基酸分析仪、核磁共振仪等进行检测，但是此类方法一方面需要昂贵的仪器投入，另一方面需要高技术的仪器专员进行操作，且前处理复杂；另一类方法是采用基于酶的生物传感器进行检测，这种方法操作简单，成本投入低，但是酶的稳定性不高以及酶的生产成本高限制了其发展。因此，建立价格低廉、操作易行、稳定快捷的生物传感方法检测精氨酸是十分重要的，而特异性识别L-精氨酸的多肽适配体才能满足需求。
发明内容
本发明旨在克服现有技术的不足，提供一种特异性识别精氨酸的多肽适配体及应用。
为了达到上述目的，本发明提供的技术方案为：
所述特异性识别精氨酸的多肽适配体的序列如SEQ ID NO.1所示：FGHIHEGY（F：苯丙氨酸、G：甘氨酸、H：组氨酸、I：异亮氨酸、E：谷氨酸、Y：酪氨酸）。
优选地，所述多肽适配体可以被提高稳定性的基团修饰，或被提供检测信号的荧光基团、同位素、电化学基团等修饰，或被亲和配基、硫醇化等修饰。
上述多肽适配体可用于制备精氨酸检测试剂盒、精氨酸检测试纸或精氨酸检测生物传感器。
下面对本发明作进一步说明：
本发明中，首先通过从蛋白库搜索精氨酸感知蛋白和转运载体，获得可与精氨酸识别的精氨酸感知蛋白的一级序列，按顺序将蛋白质一级序列切割为8个氨基酸残基，获得多肽库，将多肽库去除重复多肽序列；然后将去重后的多肽库中的多肽与L-精氨酸进行分子模拟，获得可与精氨酸特异性结合的肽适体库，优选多肽库进行试验验证；再将获得的肽适体库与精氨酸使用等温滴定量热仪进行结合验证，其验证方法为：使用10μM的多肽适配体滴定1μM的L-精氨酸，使用的溶剂和结合缓冲液为10mM的PBS缓冲液，pH为7.4，设定结合温度为37℃，从而确认可以L-精氨酸特异性结合的多肽适配体。
本发明基于分子对接结合模拟并通过等温滴定量热仪进行结合验证，发现的可与L-精氨酸特异性结合的多肽适配体序列为FGHIHEGY，其中序列从左至右为N末端至C末端，序列的字母为氨基酸的单字母简写。
本发明通过分子对接结合模拟并通过等温滴定量热仪进行结合验证，发现了可与L-精氨酸特异性结合的多肽适配体，其序列为FGHIHEGY，筛选过程比一般的噬菌体展示，合成随机多肽库等方法更为快捷方便，无需大量的人力物力投入，为L-精氨酸的快速检测，生物传感器的开发提供了稳定性好、灵敏度高、成本低、制备操作简单、修饰容易的生物识别元件。本发明获得的特异性识别精氨酸的多肽适配体相对于现有技术中的精氨酸肽适体结合力更强，更适合作为药物开发和作为检测试剂，且其合成成本低。
附图说明
图1为基于分子对接模拟预测筛选多肽适配体的流程示意图；
图2为本发明所述多肽适配体与L-精氨酸结合验证数据模拟结果图。
具体实施方式
多肽适配体与L-精氨酸的分子模拟与结合验证
A）多肽适配体与L-精氨酸的分子模拟：使用AutoDock，将L-精氨酸置于多肽适配体的活性位点，并预测其结合模式，获得L-精氨酸与多肽适配体的预测结合参数，其结合能为-3.82kcal/mol；而和L-精氨酸结构相近的其他氨基酸与多肽适配体无结合能力。
B）模拟的多肽适配体与L-精氨酸使用等温滴定量热仪进行结合验证
C）结合验证使用的多肽适配体序列为：FGHIHEGY，使用10mM的PBS配制成浓度为10μM的溶液，吸取100μL置于等温滴定量热仪的注射器中（图1），在滴定池中加入360μL的1μM的L-精氨酸进行滴定，温度设置为37℃，共滴定25滴，每滴4μL。结合验证的结果如图2，结果表明，多肽适配体与L-精氨酸结合参数Kd值为：1.334×10-5M。用等温滴定量热仪确定多肽适配体与L-精氨酸结构相近的其他氨基酸无或很弱的结合能力。
序列表
<110> 中国科学院亚热带农业生态研究所
<120> 一种特异性识别精氨酸的多肽适配体及应用
<141> 2019-07-05
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 8
<212> PRT
<213> 未知(null)
<400> 2
Phe Gly His Ile His Glu Gly Tyr
1 5

Claims (3)

1.一种特异性识别精氨酸的多肽适配体，其特征在于，所述多肽适配体的序列如SEQID NO.1所示。
2.如权利要求1所述的多肽适配体，其特征在于，所述多肽适配体被提高稳定性的基团修饰，或被提供检测信号的荧光基团、同位素、电化学基团修饰，或被亲和配基、硫醇化修饰。
3.如权利要求1或2所述的多肽适配体在制备精氨酸检测试剂盒、精氨酸检测试纸或精氨酸检测生物传感器中的应用。
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