CN112625091B - 一条结合猪瘟病毒Erns蛋白的多肽序列及应用 - Google Patents
一条结合猪瘟病毒Erns蛋白的多肽序列及应用 Download PDFInfo
- Publication number
- CN112625091B CN112625091B CN202011565041.7A CN202011565041A CN112625091B CN 112625091 B CN112625091 B CN 112625091B CN 202011565041 A CN202011565041 A CN 202011565041A CN 112625091 B CN112625091 B CN 112625091B
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- sequence
- erns protein
- affinity
- protein
- 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.)
- Active
Links
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 60
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 60
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 47
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 47
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 45
- 241000710777 Classical swine fever virus Species 0.000 title claims abstract description 29
- 238000002965 ELISA Methods 0.000 claims abstract description 12
- 238000001514 detection method Methods 0.000 claims description 10
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 4
- 238000003556 assay Methods 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 238000012986 modification Methods 0.000 claims description 3
- 230000004048 modification Effects 0.000 claims description 3
- 229960002685 biotin Drugs 0.000 claims description 2
- 235000020958 biotin Nutrition 0.000 claims description 2
- 239000011616 biotin Substances 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 239000002086 nanomaterial Substances 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- LXWYCLOUQZZDBD-LIYNQYRNSA-N csfv Chemical compound C([C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)[C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O)C1=CC=C(O)C=C1 LXWYCLOUQZZDBD-LIYNQYRNSA-N 0.000 claims 1
- 238000003032 molecular docking Methods 0.000 abstract description 13
- 206010008631 Cholera Diseases 0.000 abstract description 9
- 238000005516 engineering process Methods 0.000 abstract description 9
- 230000003993 interaction Effects 0.000 abstract description 7
- 238000012216 screening Methods 0.000 abstract description 6
- 206010037660 Pyrexia Diseases 0.000 abstract description 5
- 241000282898 Sus scrofa Species 0.000 abstract description 5
- 239000013078 crystal Substances 0.000 abstract description 3
- 238000001042 affinity chromatography Methods 0.000 abstract 1
- 239000003446 ligand Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 10
- 239000008055 phosphate buffer solution Substances 0.000 description 7
- 238000013461 design Methods 0.000 description 6
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 6
- 238000007865 diluting Methods 0.000 description 6
- 239000000872 buffer Substances 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 241000710780 Bovine viral diarrhea virus 1 Species 0.000 description 4
- 208000001726 Classical Swine Fever Diseases 0.000 description 4
- 108010067902 Peptide Library Proteins 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000010494 dissociation reaction Methods 0.000 description 4
- 230000005593 dissociations Effects 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 3
- 101710172711 Structural protein Proteins 0.000 description 3
- 238000004590 computer program Methods 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 240000003291 Armoracia rusticana Species 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 241001533384 Circovirus Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 101710121417 Envelope glycoprotein Proteins 0.000 description 1
- 101710204837 Envelope small membrane protein Proteins 0.000 description 1
- 241000710781 Flaviviridae Species 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 102100034349 Integrase Human genes 0.000 description 1
- 101710145006 Lysis protein Proteins 0.000 description 1
- 241000701076 Macacine alphaherpesvirus 1 Species 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- 206010029216 Nervousness Diseases 0.000 description 1
- 241000710778 Pestivirus Species 0.000 description 1
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 description 1
- KZIQDVNORJKTMO-WDSOQIARSA-N Trp-Arg-His Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC3=CN=CN3)C(=O)O)N KZIQDVNORJKTMO-WDSOQIARSA-N 0.000 description 1
- DZKFGCNKEVMXFA-JUKXBJQTSA-N Tyr-Ile-His Chemical compound CC[C@H](C)[C@H](NC(=O)[C@@H](N)Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O DZKFGCNKEVMXFA-JUKXBJQTSA-N 0.000 description 1
- 238000005411 Van der Waals force Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000007810 chemical reaction solvent Substances 0.000 description 1
- 238000005094 computer simulation Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 206010014599 encephalitis Diseases 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 208000021760 high fever Diseases 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 239000000710 homodimer Substances 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 201000002364 leukopenia Diseases 0.000 description 1
- 231100001022 leukopenia Toxicity 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/18—Togaviridae; Flaviviridae
- G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Pathology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Organic Chemistry (AREA)
- Virology (AREA)
- General Physics & Mathematics (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明主要涉及猪瘟病毒Erns蛋白亲和多肽及应用，该多肽由计算机模拟的虚拟分子对接技术设计而成，序列为WRHYIH。本发明以同源建模获得的猪瘟Erns蛋白晶体结构为模板，根据虚拟分子筛选技术，获得多肽序列P1。人工合成多肽后，采用酶联免疫吸附测定试验和等离子共振试验鉴定该序列与猪瘟Erns蛋白相互作用的亲和性，结果表明P1序列与猪瘟Erns蛋白具有较高的特异性和亲和力；本发明为以亲和肽作为亲和配基的亲和层析提供了可靠理论基础，可通过对多肽标记，从而对猪瘟Erns蛋白进行快速检测。
Description
技术领域
本发明主要涉及针对猪瘟病毒Erns蛋白能够特异性结合的多肽设计及应用，属于靶向猪瘟病毒Erns蛋白的亲和多肽设计及其在猪瘟病毒检测方面的应用。
背景技术
猪瘟(Classical swine fever，CSF)为猪瘟病毒(Classical swine fevervirus，CSFV)引起的一种急性、热性及高度接触性传染病。以高热、内出血、体内白细胞减少及神经症状为主要特征，呈世界性分布，被国际兽医局列为A类传染病。猪瘟病毒为黄病毒科、瘟病毒属的单股正链RNA病毒，基因组长约12.3kb，编码4个结构蛋白和7个非结构蛋白，E0蛋白又称为Erns蛋白，是CSFV的第一个囊膜糖蛋白，主要以同源二聚体的形式锚定与CSFV表面，是病毒所有结构蛋白中唯一能够分泌到细胞上清中的蛋白，能够刺激机体产生抵抗CSFV感染的中和保护抗体。
随着固相组合化学、高通量筛选技术及噬菌体表面展示等技术的发展，为大规模的构建及合成多肽库提供了有利条件，但组合化学合成肽库筛选到的多肽具有无靶向性、耗时长、反应溶剂局限性等缺点；噬菌体展示技术筛选到的肽库具有肽库容量小、肽库多样性受宿主细胞对多肽种类的选择影响、线性合成后活性消失等缺点。因此，目前大多数人选择利用基于结构模型的计算机辅助多肽设计技术，即分子对接技术。利用虚拟分子对接技术筛选多肽具有耗时短、操作简单高效、特异性强及亲和力强等优点，且具有靶向性。
发明内容
本发明通过计算机模拟的虚拟分子对接技术，设计了能够靶向结合猪瘟病毒Erns蛋白的亲和多肽，其序列为WRHYIH，命名为P1。人工固相合成P1后，通过酶联免疫吸附试验(ELISA)及等离子体共振试验(SPR)检测及鉴定其与蛋白之间的特异性及亲和力。结果表明，P1与猪瘟病毒Erns蛋白具有良好的特异性，及较高的亲和力。由此可见，由本发明设计而成的多肽序列能够用于以与猪瘟Erns蛋白结合为基础的相关研究，例如：蛋白纯化、病毒检测等。
为实现上述目的，本发明所采用的技术方案：
一条结合猪瘟病毒Erns蛋白的多肽序列为线性多肽，其序列为P1：WRHYIH。
所述多肽序列，包括以所述的P1序列为核心，任何对P1序列所进行的相应调整或修饰，修饰材料包括但不限制在生物素、亲和素、磁珠、纳米材料、荧光材料、酶类及特定的蛋白质上。
所述多肽序列，将P1序列用于任何猪瘟病毒的快速检测、鉴定，包括但不局限于酶联免疫吸附试验检测、等离子共振试验检测。
一种所述的多肽序列在制备猪瘟病毒快速检测试剂或试剂盒中的应用。
本发明的积极有益效果：
(1)本发明以同源建模的猪瘟病毒Erns蛋白为基础，利用虚拟分子对接技术设计，合成筛选出一条能够与猪瘟病毒Erns蛋白结合的多肽序列P1，该多肽具有性质稳定、结构简单、无免疫原性、易于合成及修饰，且易溶于水等优点。
(2)本发明设计出的P1序列与猪瘟病毒Erns蛋白具有良好的特异性，除与牛病毒性腹泻病毒Erns蛋白的交叉反应较高外，与其他蛋白反应较低。
(3)本发明设计出的P1序列与猪瘟病毒Erns蛋白的亲和力较高，多肽与蛋白之间相互作用的平衡解离常数KD为4.97×10-7M，即497nM，说明亲和力较好。
(4)本发明与传统噬菌体多肽筛选相比，具有操作简单，筛选强度低，研发周期短，生产成本低等优势，并可通过计算机模拟实施虚拟分子对接，实现猪瘟Erns蛋白特定位点的靶向结合，为实现对猪瘟Erns蛋白进行结构功能解析提供更好的理论支持。通过对P1序列进行标记，可实现对猪瘟病毒Erns蛋白进行定性和定量快速检测。
附图说明
图1为猪瘟病毒Erns蛋白同源建模活性口袋结构展示图。
图2为P1序列与猪瘟病毒Erns蛋白的对接结果展示图。
图3为P1序列与猪瘟病毒Erns蛋白的SPR亲和力鉴定结果。
其中，纵坐标代表传感器所检测到的信号值；横坐标代表样品在传感器中相互作用的时间。图中，从上到下各曲线对应的P1溶液浓度逐渐降低，各曲线对应浓度分别为：依次稀释成625μM、312.5μM、156.25μM、78.125μM、39μM、19.5μM。
图4为P1序列与猪瘟病毒Erns蛋白的ELISA鉴定结果。
具体实施方式
以下结合实施例对本发明的具体实施方式作进一步详细说明。
实施例1分子对接及虚拟肽库的筛选
1、Erns蛋白的准备
从PDB数据库中搜寻牛病毒性腹泻病毒Erns蛋白的晶体结构(PDB ID：4DWC)，借助计算机程序对晶体结构进行分析，选出对接活性区域(见图1)，进行分子对接。
2、虚拟多肽库的设计
借助计算机程序，采用逐个氨基酸延长的方式，以对接情况最佳的氨基酸残基为核心，再逐个增加氨基酸，实现对输入目标多肽进行批量生成，以满足分子对接计算机程序的自动调用和处理。虚拟多肽均以直链的形式生成，虚拟多肽库生成的多肽以2-9个氨基酸残基为宜。
3、对接结果的评断
分别计算多肽与蛋白结合的自由能、氢链、范德华力和疏水作用力等力学参数进行综合评定，以此来判定筛选结果，并筛选得到P1，其多肽序列为WRHYIH(SEQ ID NO.1)，其与猪瘟Erns蛋白对接的相互作用位置结果见图2。
实施例2 P1序列与人工表达Erns蛋白的亲和力鉴定
1、将人工表达纯化的猪瘟Erns蛋白用PBS液以及配套的芯片激活缓冲液稀释为250μg/mL(蛋白量计)，采用活泼酯法，先将1-(3-二甲氨基丙基)-3-乙基碳二亚胺/N-羟基琥珀酰亚胺(EDC/NHS，体积比1:1)注入装有羧基芯片的SPR检测仪中，将芯片上的羧基完全展示在表面，并活化芯片上的羧基基团，再将猪瘟Erns蛋白注入SPR检测仪中；保证EDC/NHS和蛋白分别与羧基芯片相互作用5min，实施猪瘟Erns蛋白与羧基芯片的偶联。偶联完成后，该传感芯片用于测量猪瘟Erns蛋白与P1序列之间的相互作用。
2、向传感器中注入200μLPBS缓冲液(pH 7.4)，以150μL/min的最大流速运行缓冲液，达到信号基线后，将缓冲液的运行流速降至最小，即20μL/min，以获得较为稳定的基线。
3、将合成的P1用PBS缓冲液(pH 7.4)稀释，依次稀释成625μM、312.5μM、156.25μM、78.125μM、39μM、19.5μM不同浓度的P1溶液，从低浓度到高浓度依次向传感器中注入200μL多肽溶液，运行样品时均采用20μL/min的流速，并与传感器相互作用5min，再以PBS缓冲液冲洗5min。每一个循环结束后，将250μL 0.25％SDS溶液注入传感器，用于解离剩余结合在蛋白上的P1。最终以得到的不同浓度的P1多肽溶液与猪瘟Erns蛋白相互作用的结合与解离曲线为依据，对P1序列和Erns蛋白进行亲和力分析。
结果表明，P1序列与人工表达的猪瘟Erns蛋白具有较好的亲和性结合，两者之间相互作用的平衡解离常数KD值为4.97×10-7M即497nM(见图3)。
实施例3 P1序列与人工表达Erns蛋白的ELISA鉴定
1、将人工表达纯化的猪瘟Erns蛋白用包被缓冲溶液进行稀释，以10μg/mL的浓度、每孔加入50μL进行酶标板包被，置于37℃条件下孵育2h，以PBST洗板四次后，用5％脱脂奶PBST溶液封闭2h，再以PBST洗板4次待用；以同样方式将不同病毒表达纯化的蛋白，即牛病毒性腹泻病毒Erns蛋白(BVDV-Erns)、伪狂犬病毒gD蛋白(PRV-gD)、圆环病毒Cap蛋白(PCV-Cap)、乙脑病毒E蛋白(JEV-E)，并以2％的牛血清白蛋白(BSA)进行酶标板包被作为阴性对照，PBS作为空白对照。
2、将人工固相合成并在N端生物素化修饰的P1干粉，用超纯水稀释成10mg/ml的浓度，再用PBS将多肽溶液稀释至500μg/mL，50μL/孔加入到包被好的酶标板中，混匀后置于37℃条件下孵育1h，PBST洗板4次。
3、加入以PBST稀释1000倍的辣根过氧化物酶-亲和素二抗，以50μL/孔的体积加入上述酶标板中，置于37℃条件下孵育30min，PBST洗板4次。
4、将TMB显色液以100μL/孔的体积，加入酶标板中，室温条件下，避光显色10min。
5、将2M的硫酸液终止液以50μL/孔的体积加入酶标板中，充分混匀30s后，将酶标板置于酶标仪上读取各孔在450nm处的吸光值，分析结果。
结果表明，P1序列与人工表达纯化的猪瘟Erns蛋白具有较好的亲和性与特异性，除与牛病毒性腹泻病毒Erns蛋白的交叉反应较高外，与其它病毒蛋白均不发生反应(见图4)。
序列表
<110> 河南中泽生物工程有限公司
<120> 一条结合猪瘟病毒Erns蛋白的多肽序列及应用
<130> 虚拟分子对接技术
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6
<212> PRT
<213> 人工合成()
<400> 1
Trp Arg His Tyr Ile His
1 5
Claims (4)
1.一条结合猪瘟病毒Erns蛋白的多肽，其特征在于，所述多肽为线性多肽，其序列为WRHYIH。
2.根据权利要求1所述的多肽，其特征在于，包括以所述的线性多肽为核心，对线性多肽进行的修饰，修饰材料为生物素、纳米材料、荧光材料、酶类。
3.根据权利要求1所述的多肽，其特征在于，将所述的线性多肽用于猪瘟病毒的快速检测、鉴定，包括酶联免疫吸附试验检测、等离子共振试验检测。
4.一种权利要求1所述的多肽在制备猪瘟病毒快速检测试剂或试剂盒中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011565041.7A CN112625091B (zh) | 2020-12-25 | 2020-12-25 | 一条结合猪瘟病毒Erns蛋白的多肽序列及应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011565041.7A CN112625091B (zh) | 2020-12-25 | 2020-12-25 | 一条结合猪瘟病毒Erns蛋白的多肽序列及应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112625091A CN112625091A (zh) | 2021-04-09 |
| CN112625091B true CN112625091B (zh) | 2023-03-31 |
Family
ID=75325031
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011565041.7A Active CN112625091B (zh) | 2020-12-25 | 2020-12-25 | 一条结合猪瘟病毒Erns蛋白的多肽序列及应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112625091B (zh) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106496304A (zh) * | 2016-11-15 | 2017-03-15 | 河南省农业科学院 | 与猪瘟病毒e2蛋白特定区域结合多肽配基设计及应用 |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2202298A1 (en) * | 2008-12-23 | 2010-06-30 | Stichting Dienst Landbouwkundig Onderzoek | Recombinant classical swine fever virus (CSFV) comprising a modified E2 protein and methods for generating said recombinant CSFV |
| KR101317417B1 (ko) * | 2011-03-11 | 2013-10-10 | 베트올 (주) | 돼지열병바이러스의 탐지용 항원, 이를 이용한 항체 검출 방법, 및 이를 포함하는 탐지키트 |
| CN104650185B (zh) * | 2015-02-14 | 2017-11-10 | 河南省农业科学院 | 一条与猪瘟e0蛋白相结合的多肽序列及其应用 |
| CN106749520B (zh) * | 2016-11-15 | 2020-04-14 | 河南省农业科学院 | 针对猪瘟病毒e2蛋白高亲和性多肽序列的设计及应用 |
| CN108383895B (zh) * | 2018-05-03 | 2021-04-27 | 河南省农业科学院 | 一条可与猪瘟病毒e2蛋白结合的亲和肽及其应用 |
-
2020
- 2020-12-25 CN CN202011565041.7A patent/CN112625091B/zh active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106496304A (zh) * | 2016-11-15 | 2017-03-15 | 河南省农业科学院 | 与猪瘟病毒e2蛋白特定区域结合多肽配基设计及应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112625091A (zh) | 2021-04-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20200309770A1 (en) | Chemiluminescence immunoassay kit for adiponectin, and preparation method and use thereof | |
| CN106749520B (zh) | 针对猪瘟病毒e2蛋白高亲和性多肽序列的设计及应用 | |
| CN112782156B (zh) | 一种壳多糖酶3样蛋白1试剂盒及其制备方法 | |
| CN103197081B (zh) | 一种蛋白质芯片及其制备和检测方法 | |
| CN109444434A (zh) | 双抗原夹心检测抗体的方法 | |
| Wang et al. | Target protein induced cleavage of a specific peptide for prostate-specific antigen detection with positively charged gold nanoparticles as signal enhancer | |
| KR20030096005A (ko) | 면역학적 측정방법, 면역학적 측정용 시약 및 그의 제조방법 | |
| CN114594262A (zh) | 基于双功能融合蛋白的真菌毒素磁化学发光免疫分析试剂盒及其应用 | |
| CN105044355B (zh) | 用于检测EB病毒Rta/IgG抗体的化学发光试剂盒及其应用 | |
| CN112462061A (zh) | 检测h1n1、rsv-a和adv3的试剂盒及其应用 | |
| CN112707948B (zh) | 一条结合猪瘟病毒e0蛋白的多肽序列及应用 | |
| CN112625091B (zh) | 一条结合猪瘟病毒Erns蛋白的多肽序列及应用 | |
| CN111537483B (zh) | 检测冈田酸的荧光核酸适配体传感器、其制备方法与使用该传感器检测冈田酸的方法 | |
| CN108383895B (zh) | 一条可与猪瘟病毒e2蛋白结合的亲和肽及其应用 | |
| CN112375754A (zh) | 基于人血管紧张素转化酶2的严重急性呼吸系统综合症冠状病毒2亲和多肽 | |
| JP4975601B2 (ja) | Ｈｃｖ抗体測定用試薬キット及びｈｃｖ抗体測定方法 | |
| EP1978365A1 (en) | Reagent kit and method for measuring HCV antibody | |
| CN105181956B (zh) | 基于金属离子特异响应的荧光检测法在免疫检测中的应用 | |
| CN106518969B (zh) | 与脂肪酶靶向结合的肽配基序列及其应用 | |
| JP6519478B2 (ja) | 抗ｈｔｌｖ抗体の免疫測定における非特異反応の低減方法 | |
| CN107884371A (zh) | 用于高通量抗体快速检测的荧光素酶免疫吸附方法 | |
| WO2021003945A1 (zh) | 特异性识别精氨酸的多肽适配体及应用 | |
| CN106749519B (zh) | 基于计算机模拟的csfv e2蛋白靶向结合的肽配基序列设计及应用 | |
| CN110702925A (zh) | 同时检测csfv、ppv、jev、prrsv抗体的荧光标记蛋白芯片制备和检测方法 | |
| CN109917124A (zh) | 一种丙型肝炎病毒抗原抗体联合检测试剂盒 |
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 | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20230302 Address after: No. 218, Ping'an Avenue, Zhengdong New District, Zhengzhou City, Henan Province, 450000 Applicant after: Longfor Modern Immunology Laboratory Address before: 450000 floors 1-2, building 4, south of Yingbin Road, HANGGANG District, Zhengzhou City, Henan Province Applicant before: HENAN ZHONGZE BIOLOGICAL ENGINEERING CO.,LTD. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |