CN110257388A - 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

Info

Publication number
CN110257388A
CN110257388A CN201910604881.0A CN201910604881A CN110257388A CN 110257388 A CN110257388 A CN 110257388A CN 201910604881 A CN201910604881 A CN 201910604881A CN 110257388 A CN110257388 A CN 110257388A
Authority
CN
China
Prior art keywords
arginine
polypeptide aptamers
aptamers
polypeptide
specific recognition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201910604881.0A
Other languages
English (en)
Other versions
CN110257388B (zh
Inventor
冯泽猛
贺玉敏
邓磊
曹忠
肖忠良
印遇龙
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Subtropical Agriculture of CAS
Original Assignee
Institute of Subtropical Agriculture of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute of Subtropical Agriculture of CAS filed Critical Institute of Subtropical Agriculture of CAS
Priority to CN201910604881.0A priority Critical patent/CN110257388B/zh
Publication of CN110257388A publication Critical patent/CN110257388A/zh
Priority to US17/052,173 priority patent/US11774455B2/en
Priority to PCT/CN2019/119036 priority patent/WO2021003945A1/zh
Application granted granted Critical
Publication of CN110257388B publication Critical patent/CN110257388B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6806—Determination of free amino acids
    • G01N33/6812—Assays for specific amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Plant Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

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。
CN201910604881.0A 2019-07-05 2019-07-05 一种特异性识别精氨酸的多肽适配体及应用 Active CN110257388B (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CN201910604881.0A CN110257388B (zh) 2019-07-05 2019-07-05 一种特异性识别精氨酸的多肽适配体及应用
US17/052,173 US11774455B2 (en) 2019-07-05 2019-11-16 Peptide aptamer for specific recognition of arginine and its application
PCT/CN2019/119036 WO2021003945A1 (zh) 2019-07-05 2019-11-16 特异性识别精氨酸的多肽适配体及应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910604881.0A CN110257388B (zh) 2019-07-05 2019-07-05 一种特异性识别精氨酸的多肽适配体及应用

Publications (2)

Publication Number Publication Date
CN110257388A true CN110257388A (zh) 2019-09-20
CN110257388B CN110257388B (zh) 2023-01-03

Family

ID=67924759

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910604881.0A Active CN110257388B (zh) 2019-07-05 2019-07-05 一种特异性识别精氨酸的多肽适配体及应用

Country Status (3)

Country Link
US (1) US11774455B2 (zh)
CN (1) CN110257388B (zh)
WO (1) WO2021003945A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021003945A1 (zh) * 2019-07-05 2021-01-14 中国科学院亚热带农业生态研究所 特异性识别精氨酸的多肽适配体及应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150292005A1 (en) * 2012-04-27 2015-10-15 Kirin Kabushiki Kaisha Nucleic acid molecules for highly sensitive detection of ligands, screening method for nucleic acid molecules, and optimization method for sensitivity of nucleic acid molecules
CN106518970A (zh) * 2016-11-15 2017-03-22 郑州大学第附属医院 可与甲胎蛋白特异结合的多肽序列及其应用
CN108197429A (zh) * 2018-01-03 2018-06-22 中国科学院亚热带农业生态研究所 一种基于分子对接技术的代谢物肽适体快速筛选方法

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101912826B1 (ko) 2016-10-25 2018-10-30 한국생산기술연구원 특정분자 친화력을 갖는 하이드로겔 및 이를 이용한 하이드로겔의 부피 조절 방법
CN110257388B (zh) 2019-07-05 2023-01-03 中国科学院亚热带农业生态研究所 一种特异性识别精氨酸的多肽适配体及应用
CN110220960B (zh) 2019-07-05 2024-02-06 长沙理工大学 一种l-精氨酸的检测方法及传感器
CN110220961B (zh) 2019-07-05 2024-01-12 长沙理工大学 一种基于多肽复合膜修饰电极的l-精氨酸检测方法及传感器
CN110426435B (zh) 2019-07-05 2021-10-19 中国科学院亚热带农业生态研究所 一种基于肽适体的精氨酸生物传感器及其制备方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150292005A1 (en) * 2012-04-27 2015-10-15 Kirin Kabushiki Kaisha Nucleic acid molecules for highly sensitive detection of ligands, screening method for nucleic acid molecules, and optimization method for sensitivity of nucleic acid molecules
CN106518970A (zh) * 2016-11-15 2017-03-22 郑州大学第附属医院 可与甲胎蛋白特异结合的多肽序列及其应用
CN108197429A (zh) * 2018-01-03 2018-06-22 中国科学院亚热带农业生态研究所 一种基于分子对接技术的代谢物肽适体快速筛选方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
YUMIN HE等: "An electrochemical impedimetric sensing platform based on a peptide aptamer identified by high-throughput molecular docking for sensitive l-arginine detection", 《BIOELECTROCHEMISTRY》 *
袁海燕等: "核酸适配体为荧光探针对精氨酸的手性识别", 《光谱学与光谱分析》 *
高婕: "基于适配体的酶分子检测新方法", 《中国优秀硕士学位论文全文数据库 基础科学辑》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021003945A1 (zh) * 2019-07-05 2021-01-14 中国科学院亚热带农业生态研究所 特异性识别精氨酸的多肽适配体及应用
US11774455B2 (en) 2019-07-05 2023-10-03 Institute Of Subtropical Agriculture, Chinese Academy Of Sciences Peptide aptamer for specific recognition of arginine and its application

Also Published As

Publication number Publication date
WO2021003945A1 (zh) 2021-01-14
US20220260582A1 (en) 2022-08-18
CN110257388B (zh) 2023-01-03
US11774455B2 (en) 2023-10-03

Similar Documents

Publication Publication Date Title
Krokhin Sequence-specific retention calculator. Algorithm for peptide retention prediction in ion-pair RP-HPLC: application to 300-and 100-Å pore size C18 sorbents
CN105143872B (zh) 通过质谱分析用复用内标物对蛋白质和蛋白质修饰的绝对定量
La Mendola et al. Probing the copper (II) binding features of angiogenin. Similarities and differences between a N-terminus peptide fragment and the recombinant human protein
US20100331199A1 (en) Method for the detection and/or enrichment of analyte proteins and/or analyte peptides from a complex protein mixture
CN110231490A (zh) 一种探针及其应用
Mielczarek et al. Hemorphins—From discovery to functions and pharmacology
CN110426435A (zh) 一种基于肽适体的精氨酸生物传感器及其制备方法
US7186799B2 (en) Peptide and amine examination method using the same
CN110257388A (zh) 一种特异性识别精氨酸的多肽适配体及应用
Ge et al. Membrane topology of rat sodium-coupled neutral amino acid transporter 2 (SNAT2)
US20040242854A1 (en) Chemical capture reagent
CN103255109B (zh) 杂交瘤细胞株afg1g6、其单克隆抗体及黄曲霉毒素g1免疫层析试纸条
CN105988001A (zh) 一种测定非对称二甲基精氨酸浓度的试剂盒及方法
CN107290552B (zh) 高凝血状态的生物标志物及其应用
US20080319678A1 (en) Mass Tagging for Quantitative Analysis of Biomolecules using 13C Labeled Phenylisocyanate
Chandra et al. Design of peptide ligands for affinity purification of human growth hormone
Gunzburg et al. Use of SPR to Study the Interaction of G7-18NATE Peptide with the Grb7-SH2 Domain
CN107406515A (zh) 合成的双表位化合物
Kawabata et al. Peptide-based immunoadsorbents: Molecular grafting of IgG–Fc-binding epitopes of Protein A onto a de novo-designed helix-loop-helix peptide
CN106995491A (zh) 新型澳洲坚果过敏原
RU2010133156A (ru) Калибратор/регулятор для синхронного анализа белков, способных к комплексообразованию друг с другом
CN110498838B (zh) 用于fpgs和ggh蛋白表达量检测的特征性肽段及其应用
CN114933638B (zh) 特异性靶向cd28的多元环肽配体框架
Torrini et al. Advances and perspectives in the analytical technology for small peptide hormones analysis: a glimpse to gonadorelin
US20080009070A1 (en) Amine detecting method

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant