CN114656561A - 一种抗ip-10单克隆抗体及其制备方法和应用 - Google Patents

一种抗ip-10单克隆抗体及其制备方法和应用 Download PDF

Info

Publication number
CN114656561A
CN114656561A CN202210211321.0A CN202210211321A CN114656561A CN 114656561 A CN114656561 A CN 114656561A CN 202210211321 A CN202210211321 A CN 202210211321A CN 114656561 A CN114656561 A CN 114656561A
Authority
CN
China
Prior art keywords
variable region
monoclonal antibody
thr
chain variable
ser
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
CN202210211321.0A
Other languages
English (en)
Other versions
CN114656561B (zh
Inventor
陈雨欣
沈瀚
陈琳
耿毓
陶月
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.)
Nanjing Drum Tower Hospital
Original Assignee
Nanjing Drum Tower Hospital
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 Nanjing Drum Tower Hospital filed Critical Nanjing Drum Tower Hospital
Priority to CN202210211321.0A priority Critical patent/CN114656561B/zh
Publication of CN114656561A publication Critical patent/CN114656561A/zh
Application granted granted Critical
Publication of CN114656561B publication Critical patent/CN114656561B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • 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
    • C12N2510/00—Genetically modified cells
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/521—Chemokines
    • G01N2333/522—Alpha-chemokines, e.g. NAP-2, ENA-78, GRO-alpha/MGSA/NAP-3, GRO-beta/MIP-2alpha, GRO-gamma/MIP-2beta, IP-10, GCP-2, MIG, PBSF, PF-4 or KC
    • 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)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Hematology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Physics & Mathematics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Oncology (AREA)
  • Plant Pathology (AREA)
  • Communicable Diseases (AREA)
  • Virology (AREA)
  • Pain & Pain Management (AREA)

Abstract

本发明公开了一种抗IP‑10单克隆抗体及其制备方法和应用，公开了抗IP‑10单克隆抗体、抗体的制备方法、抗体在制备用于多种疾病中药物的应用，还公开了本发明提供的抗体可配对使用检测抗原的应用。本发明抗IP‑10单克隆抗体能特异性地与IP‑10抗原或sIP‑10抗原结合，为通过基因工程方法诊断或治疗或预防病毒感染、肿瘤或炎性疾病建立基础；本发明抗体还可配对使用，精准测量检测血清中的sIP‑10浓度；此外本发明所述的抗IP‑10单克隆抗体具有特异性抗原结合结构域，可靶向结合IP‑10蛋白，也能够用于免疫组化、ELISA等试验。

Description

一种抗IP-10单克隆抗体及其制备方法和应用
技术领域
本发明属于生物医药和基因工程技术领域，具体涉及一种抗IP-10单克隆抗体及其制备方法和应用。
背景技术
趋化因子IP-10(interferon inducible protein-10)，也称为CXCL10，它归类于CXC趋化因子，受体是CXCR3，分子量是10kD。IP-10来源于活化的成纤维细胞、单核巨噬细胞、内皮细胞和各种淋巴细胞等30多种细胞。具有强大的招募中性粒细胞,促进多种细胞因子分泌及抑制部分肿瘤生长等多种生物学作用。IP-10和CXCL11/I-IAC关系密切，且可相互转化。在诸如病毒感染的炎症反应时，各系统中(如神经系统、肝脾等)IP-10的浓度可出现不同程度的增高，在移植排斥反应中同样有明显升高。目前发现其与多种疾病有关，如常见的皮肤疾病：变应性接触性皮炎，银屑病，系统性红斑狼疮，尖锐湿疣等，还有结核，SARS，病毒性肝炎等。在诸如病毒感染的炎症反应时，各系统中(如神经系统、肝脾等)IP-10的浓度可出现不同程度的增高，在移植排斥反应中同样有明显升高，比如在肾脏移植术中Matz等发现在手术后IP-10的mRNA和蛋白质水平都升高了(Matz M等，Kidney Int,2006)。
目前，开发IP-10在临床的上诊断、治疗作用，以阻止肿瘤、病毒、炎症等某些疾病的临床进程，或者通过IP-10的靶向治疗达到减弱器官移植排斥中的免疫反应，具有十分重要的意义。
发明内容
有鉴于此，本发明期望提供一种抗IP-10单克隆抗体及其制备方法和应用，所提供的抗IP-10单克隆抗体可配对使用，可通过双抗体夹心ELISA法实现对重组IP-10抗原和临床样本中sIP-10的精确检测。
为达到上述目的，本发明的技术方案是这样实现的：
一种抗IP-10单克隆抗体，包括重链可变区(VH)和轻链可变区(VL)；
所述重链可变区的氨基酸序列如SEQ ID NO:1所示；
所述轻链可变区的氨基酸序列如SEQ ID NO:3所示。
进一步地，编码上述抗IP-10单克隆抗体，其DNA序列包括重链可变区和轻链可变区的编码DNA序列；
所述重链可变区的编码DNA序列如SEQ ID NO:2所示；
所述轻链可变区的编码DNA序列如SEQ ID NO:4所示。
进一步地，另外一种抗IP-10单克隆抗体，包括重链可变区(VH)和轻链可变区(VL)；
所述重链可变区的氨基酸序列如SEQ ID NO:5所示；
所述轻链可变区的氨基酸序列如SEQ ID NO:7所示。
进一步地，编码上述抗IP-10单克隆抗体，其DNA序列包括重链可变区和轻链可变区的编码DNA序列；
所述重链可变区的编码DNA序列如SEQ ID NO:6所示；
所述轻链可变区的编码DNA序列如SEQ ID NO:8所示。
这里，上述抗IP-10单克隆抗体，能特异性地与IP-10抗原或sIP-10抗原结合，为通过基因工程方法诊断或治疗或预防病毒感染、肿瘤或炎性疾病建立基础；且上述抗IP-10单克隆抗体可配对使用，精准测量检测血清中的sIP-10浓度，可快速帮助医生判断患者的免疫状态。
本发明还另外提供一种抗IP-10单克隆抗体的制备方法，制备上述抗IP-10单克隆抗体，包括以下步骤：
1)用人IP-10胞外免疫兔，作出免疫反应后，处死，取脾脏，分离获得脾脏细胞；
2)筛选获得能特异性结合人IP-10的B细胞；
3)将B细胞进行亚克隆，获得抗体重链和轻链的可变区编码序列；
4)获得的可变区编码序列进行重组、转染、纯化后获得抗IP-10单克隆抗体。
这里，上述可变区编码序列为RNA序列；此外，本发明方法提供了一款IP-10兔来源单克隆抗体，丰富了抗体的类型。
进一步地，本发明还提供药物组合物，包括上述抗IP-10单克隆抗体和药学上可接受的载体。
这里，所述药物组合物包括抗IP-10药物、干扰素、抗IP-10单克隆抗体、抗IP-10多克隆抗体、核苷类似物、DNA聚合酶抑制剂、siRNA药物或治疗性疫苗等。
进一步地，本发明还提供一种表达载体，包含上述抗IP-10单克隆抗体的编码DNA，分别用于表达上述抗IP-10单克隆抗体。
进一步地，本发明还提供一种原核或真核宿主细胞，包含上述表达载体。
进一步地，本发明还提供抗IP-10单克隆抗体在制备治疗或预防人病毒感染、肿瘤及炎性疾病药物中的用途。
进一步地，本发明还提供一种用于检测IP-10抗原或sIP-10抗原的试剂盒，包括上述抗IP-10单克隆抗体。
这里，上述抗IP-10单克隆抗体具有特异性抗原结合结构域，可靶向结合IP-10蛋白，也能够用于免疫组化、ELISA等试验。
进一步地，本发明还提供一种检测IP-10抗原或sIP-10抗原的方法，使用上述用于检测IP-10抗原或sIP-10抗原的试剂盒，通过双抗体夹心ELISA法进行检测。
本发明有益效果如下：1)本发明提供一种抗IP-10单克隆抗体及其制备方法和应用，能特异性地与IP-10抗原或sIP-10抗原结合，为通过基因工程方法诊断或治疗或预防病毒感染、肿瘤或炎性疾病建立基础；2)本发明提供的抗IP-10单克隆抗体可配对使用，精准测量检测血清中的sIP-10浓度，可快速帮助医生判断患者的免疫状态；3)本发明提供了一款IP-10兔来源单克隆抗体，丰富了抗体的类型；4)本发明所述的抗IP-10单克隆抗体具有特异性抗原结合结构域，可靶向结合IP-10蛋白，也能够用于免疫组化、ELISA等试验。
附图说明
图1为本发明实施例1中免疫后的新西兰白兔的血清效价检测结果图；
图2为本发明实施例3中纯化的单克隆抗体特异性结合IP-10结果图；
图3为本发明实施例4中双抗体夹心法检测重组蛋白IP-1的ELISA分析结果。
具体实施方式
为了能够更加详尽地了解本发明的特点与技术内容，下面结合附图对本发明的实现进行详细阐述，所附附图仅供参考说明之用，并非用来限定本发明。除非另有说明，本发明所用的技术和科学术语与本发明所属领域的普通技术员通常所理解的含义相同。除非另有说明，下文描述的实施例的方法和材料均可以通过市场购买获得的常规产品。本发明所属领域技术员将会理解，下文描述的方法和材料，仅是示例性的，而不应视为限定本发明的范围。
实施例1：IP-10特异性单克隆抗体的制备
1)用重组表达的人IP-10胞外区免疫新西兰大白兔，获得针对人IP-10的免疫反应。
抗原采用人IP-10胞外结构域的重组蛋白(IP-10)。第0天用含有400μg IP-10蛋白的400μl的弗式完全佐剂(Sigma-Aldrich)的1：1乳液皮下免疫新西兰白兔。随后，第7，21和42天皮下注射含有200μg IP-10蛋白的弗式不完全佐剂(Sigma-Aldrich)的1：1乳液，从而对新西兰白兔(#R6586、6587)进行加强免疫。免疫后的新西兰白兔血清效价在三次免疫后达到104后以上。图1为本发明实施例1中免疫后的新西兰白兔的血清效价检测结果图，如图1所示，表现最高抗体滴度的兔子(#R6586)接受了200μg IP-10(不含佐剂)静脉注射加强免疫。
2)筛选得特异性结合人IP-10的淋巴B细胞，并进行亚克隆。
使用Lighting-Link R-Phycoerythrin(R-PE)Conjugation Kit(InnovaBiosciences公司)标记IP-10。将IP-10浓度调整至不超过1mg/ml；加1ul的LL-modifier试剂至10μl IP-10中混匀；将混合物加入Lighting-Link mix中的干粉状物中，重悬粉状物；室温放置至少3小时或者过夜；向混合物中加入1μl的LL-quencher试剂，30分钟后R-PE标记的IP-10即可使用。
提取脾脏并进行均质化以产生单细胞悬液，并加入荧光标记抗体：①PE-Cy7标记抗兔IgG抗体5μl；②APC标记抗兔MHCII抗体5μl；③R-PE标记IP-10 2ul。振荡混匀；用流式细胞仪分选收集MHCII-IgG+细胞，即为分泌IP-10抗体的B细胞。
3)IP-10特异性B细胞亚克隆。
使用RNA提取试剂盒Neasy mini Kit(Qiagen)提取分泌IP-10抗体的B细胞的RNA。采用SuperScriptⅢOne-Step RT-PCR System with Platinum Taq DNA Polymerase(Invitrogen公司)进行RT-PCR反应，使用PrimerPremier5软件分别设计扩增兔单克隆抗体的重链和轻链全长基因的RT-PCR引物，重链引物序列分别为：RHC1、RHC2；轻链引物序列分别为：RLC1、RLC2，将特异性B细胞的RNA逆转录为cDNA，分别扩增编码抗体重链和轻链的全长片段。
其中，RT-PCR引物序列分别如下：
RHC1：5’-CCGTCCAAGCTTATGGAGACTGGGCTGCGCTGGC-3’
RHC2：5’-CAACAAGGATCCCTATTTACCCGGAGAGCGGGAG-3’
RLC1：5’-CCGTCCAAGCTTATGGACACGAGGGCCCCCACTC-3’
RLC2：5’-CAACAAGGATCCCTAACAGTCACCCCTATTGAAGC-3’
反应条件为50℃30min，94℃2min，随后进行(94℃30s，57℃30s，68℃1min)35次循环，68℃延伸5min，4℃5min。PCR扩增后，将PCR产物经琼脂糖凝胶电泳回收纯化。
实施例2：编码IP-10单克隆抗体重链和轻链全长基因测序及抗体重组生产
(1)编码IP-10单克隆抗体重链和轻链全长基因测序
将克隆获得的编码全长重链与轻链基因的PCR产物连接到pcDNA3.1(ThermoFisher Scientific)表达载体上，并将连接产物转化DH5α感受态细菌中，在含有氨苄青霉素的平板上37℃培养过夜，随机挑取10个单菌落用RT-PCR引物进行扩增，RT-PCR引物序列参照实施例中RHC1、RHC2、RLC1、RLC2，反应条件为：94℃预变性30s，(94℃变性30s，57℃退火30s，68℃延伸1min)30次循环，最后68℃延伸5min。取5ul PCR产物在1％琼脂糖凝胶上进行电泳检测，在阳性转化子中鉴定出还有抗体重链和轻链的转化子。同时将阳性转化子送至南京擎科公司测序，最终获得1C5和10D5的独特核苷酸/蛋白序列，序列信息分别如下：
1C5重链可变区氨基酸序列：SEQ ID NO.1
METGLRWLLLVAVLKGVQCQEQLEESGGDLVKPEGSLTLTCKASGFTISNLYYYMCWVRQAPGKGLEWIACIYTGSDDSSEYASWAKGRFTISKSSSTTVTLQMTSLTAADTATYFCARQNGGPFDLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK
1C5重链可变区DNA序列：SEQ ID NO.2
ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGGAGCAGCTGGAGGAGTCCGGGGGAGACCTGGTCAAGCCTGAGGGATCCCTGACACTCACCTGCAAAGCCTCTGGATTCACCATCAGTAATCTTTATTACTACATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGTATTTATACAGGCAGTGATGATAGTAGTGAGTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAAGCTCGTCGACCACGGTGACTCTGCAAATGACCAGTCTGACTGCCGCGGACACGGCCACCTATTTCTGTGCGAGACAGAATGGTGGCCCTTTTGACTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGCTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCATGTGCCCACCCCCTGAACTCCCGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGACCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATAG
1C5轻链可变区氨基酸序列：SEQ ID NO.3
MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSSVSAAVGGTVTINCQASQTLYNNKNLAWYQQKPGQPPKLLIYGTSSLASGVPSRFRGSGSGTQFTLTISDLECDDAAAYYCQGEFSCGSADCFAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC
1C5轻链可变区DNA序列：SEQ ID NO.4
ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAGTGCTGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAACTGCCAGGCCAGTCAGACTCTTTATAATAACAAAAATTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATGGTACATCCAGTCTGGCATCTGGGGTCCCATCGCGGTTCAGAGGCAGTGGATCTGGGACACAATTCACTCTCACCATCAGCGACCTGGAGTGTGACGATGCTGCCGCTTATTATTGTCAAGGCGAATTTAGTTGTGGTAGTGCTGATTGTTTTGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG
10D5重链可变区氨基酸序列：SEQ ID NO.5
METGLRWLLLVAVLKGVQCQEQLVESGGGLVQPEGSLTLTCKASGFDFSSNVMCWVRQAPGKGLEWIACIGAGSGGDTYCARWAKGRFTISKTSPTTVTLQMTSLTAADTASYFCTSRGDGVDPYDLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK
10D5重链可变区DNA序列：SEQ ID NO.6
ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGGAGCAGCTGGTGGAGTCCGGGGGAGGCCTGGTCCAGCCTGAGGGATCCCTGACACTCACCTGCAAAGCCTCTGGATTCGACTTCAGTAGCAATGTAATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAATGGATCGCATGCATTGGCGCTGGTAGTGGTGGTGATACTTACTGCGCGAGGTGGGCGAAGGGCCGATTCACCATCTCCAAAACCTCGCCGACCACGGTGACTCTGCAAATGACCAGTCTGACAGCCGCGGACACGGCCTCCTATTTCTGTACGAGTAGGGGTGATGGTGTTGATCCTTATGACTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGCTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCATGTGCCCACCCCCTGAACTCCCGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGACCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATAG
10D5轻链可变区氨基酸序列：SEQ ID NO.7
MDTRAPTQLLGLLLLWLPGATFAQVLTQTASPVSAAVGGTVTINCQASQSVYSNNYLSWFQQKPGQPPKQLIYDASTLASGVPSRFKGSGSGTQFTLTITDVLCDDAATYYCLGGYDCSSADCWAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC
10D5轻链可变区DNA序列：SEQ ID NO.8
ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAGTGCTGACCCAGACTGCATCCCCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAACTGCCAGGCCAGTCAGAGTGTTTATAGTAACAATTACTTATCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAGCAACTGATCTATGATGCATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCACCGACGTACTGTGTGACGATGCTGCCACTTACTACTGTCTAGGCGGTTATGATTGTAGTAGTGCTGATTGTTGGGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG
(2)生产和纯化IP-10抗体
将表达单克隆抗体重链和轻链的质粒共转染293F细胞，并在37℃摇瓶中培养4天，采用Protein A亲和层析柱从细胞培养上清中分离纯化目的抗体。
实施例3:ELISA检测单克隆抗体1C5和10D5对IP-10蛋白的结合能力
在96孔板(Costar，42592)中包被100μl IP-10蛋白(1μg/mL)4℃冰箱过夜。次日用200μl 0.1％Triton-X的PBS洗涤液洗涤5遍，在200μl含有5％奶粉的PBS-T(0.05％吐温)中室温封闭1小时，洗涤5遍，加入倍比稀释的IP-10抗体1C5或10D5分别100μl室温孵育1小时，洗涤5遍，每孔中加入100μl耦合辣根过氧化物酶的山羊抗兔IgG于室温孵育1小时。将板用0.1％Triton-X的PBS洗涤液洗涤五次，然后每孔中依次加入TMB显色液，显色3分钟，加入25ul终止液使反应停止，轻轻振荡混匀。设定全自动多功能酶标仪双波长为450nm/610nm，用空白孔调零点后测定各孔OD值。图2为本发明实施例3中纯化的单克隆抗体特异性结合IP-10结果图，如图2所示，纯化后的单克隆抗体能够特异性结合IP-10蛋白。随着抗体浓度的增加，OD读数也呈显著增加的趋势。
实施例4:双抗体夹心法ELISA检测IP-10浓度
(1)生物素标记抗体的制备
利用EZ-LinkTM Sulfo-NHS-LC-Biotin试剂盒(Thermo Scientific)标记检测抗体10D5。具体方法如下：将sulfo-NHS-LC-biotin从冰箱中取出，并平衡至室温。每1mg IgG抗体加入26.6μl生物素标记试剂，置于冰上2小时。标记结束后，通过透析方法除去多余的未标记上的生物素试剂。
(2)双抗体夹心ELISA试验
在96孔板(Costar)中加入100μl用碳酸盐缓冲溶液(PH＝9.4)稀释后的捕获的抗体1C5(1μg/mL)铺板，在4℃孵育过夜。次日用200μl 0.1％Triton-X的PBS洗涤液洗涤5遍；并在200μl含有5％奶粉的PBS-T(0.05％吐温)中室温封闭1小时，洗涤5遍；加入100μl等比稀释的重组蛋白IP-10，室温孵育1小时；随后用洗涤液洗板5遍，每孔中加入在(1)中生物素标记的检测抗体10D5(1μg/mL)，室温孵育1小时；随后用洗涤液洗板5遍，每孔加入100μl辣根过氧化物酶－链霉亲和素(HRP-streptavidin，Jackson Immuno Lab)，并在室温孵育1小时。将板用洗涤液清洗五次，然后每孔中依次加入TMB显色液，显色3分钟，加入25ul终止液使反应停止，轻轻振荡混匀。设定全自动多功能酶标仪双波长为450nm/610nm，用空白孔调零点后测定各孔OD值。
图3为本发明实施例4中双抗体夹心法检测重组蛋白IP-1的ELISA分析结果，从图3可知，本发明提供的可配对使用的抗IP-10单克隆抗体利用双抗体夹心法检测重组蛋白IP-10的浓度可低至1.2ng/ml，检测精度高。
以上所涉及试剂、仪器的具体货号及型号不作限制及详细描述，作为公知常识，本领域的技术人员能够理解。
以上所述，仅为本发明的较佳实施例而已，并非用于限定本发明的保护范围，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
序列表
<110> 南京鼓楼医院
<120> 一种抗IP-10单克隆抗体及其制备方法和应用
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 461
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly
1 5 10 15
Val Gln Cys Gln Glu Gln Leu Glu Glu Ser Gly Gly Asp Leu Val Lys
20 25 30
Pro Glu Gly Ser Leu Thr Leu Thr Cys Lys Ala Ser Gly Phe Thr Ile
35 40 45
Ser Asn Leu Tyr Tyr Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys
50 55 60
Gly Leu Glu Trp Ile Ala Cys Ile Tyr Thr Gly Ser Asp Asp Ser Ser
65 70 75 80
Glu Tyr Ala Ser Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Ser Ser
85 90 95
Ser Thr Thr Val Thr Leu Gln Met Thr Ser Leu Thr Ala Ala Asp Thr
100 105 110
Ala Thr Tyr Phe Cys Ala Arg Gln Asn Gly Gly Pro Phe Asp Leu Trp
115 120 125
Gly Pro Gly Thr Leu Val Thr Val Ser Ser Gly Gln Pro Lys Ala Pro
130 135 140
Ser Val Phe Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr
145 150 155 160
Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr
165 170 175
Val Thr Trp Asn Ser Gly Thr Leu Thr Asn Gly Val Arg Thr Phe Pro
180 185 190
Ser Val Arg Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Ser
195 200 205
Val Thr Ser Ser Ser Gln Pro Val Thr Cys Asn Val Ala His Pro Ala
210 215 220
Thr Asn Thr Lys Val Asp Lys Thr Val Ala Pro Ser Thr Cys Ser Lys
225 230 235 240
Pro Met Cys Pro Pro Pro Glu Leu Pro Gly Gly Pro Ser Val Phe Ile
245 250 255
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
260 265 270
Val Thr Cys Val Val Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln
275 280 285
Phe Thr Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro
290 295 300
Leu Arg Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu
305 310 315 320
Pro Ile Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys
325 330 335
Val His Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
340 345 350
Ala Arg Gly Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro
355 360 365
Arg Glu Glu Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn
370 375 380
Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys
385 390 395 400
Ala Glu Asp Asn Tyr Lys Thr Thr Pro Thr Val Leu Asp Ser Asp Gly
405 410 415
Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln
420 425 430
Arg Gly Asp Val Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn
435 440 445
His Tyr Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys
450 455 460
<210> 2
<211> 1386
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60
gagcagctgg aggagtccgg gggagacctg gtcaagcctg agggatccct gacactcacc 120
tgcaaagcct ctggattcac catcagtaat ctttattact acatgtgctg ggtccgccag 180
gctccaggga aggggctgga gtggatcgca tgtatttata caggcagtga tgatagtagt 240
gagtacgcga gctgggcgaa aggccgattc accatctcca aaagctcgtc gaccacggtg 300
actctgcaaa tgaccagtct gactgccgcg gacacggcca cctatttctg tgcgagacag 360
aatggtggcc cttttgactt gtggggccca ggcaccctgg tcaccgtctc ctcagggcaa 420
cctaaggctc catcagtctt cccactggcc ccctgctgcg gggacacacc cagctccacg 480
gtgaccctgg gctgcctggt caaaggctac ctcccggagc cagtgaccgt gacctggaac 540
tcgggcaccc tcaccaatgg ggtacgcacc ttcccgtccg tccggcagtc ctcaggcctc 600
tactcgctga gcagcgtggt gagcgtgacc tcaagcagcc agcccgtcac ctgcaacgtg 660
gcccacccag ccaccaacac caaagtggac aagaccgttg cgccctcgac atgcagcaag 720
cccatgtgcc caccccctga actcccgggg ggaccgtctg tcttcatctt ccccccaaaa 780
cccaaggaca ccctcatgat ctcacgcacc cccgaggtca catgcgtggt ggtggacgtg 840
agccaggatg accccgaggt gcagttcaca tggtacataa acaacgagca ggtgcgcacc 900
gcccggccgc cgctacggga gcagcagttc aacagcacga tccgcgtggt cagcaccctc 960
cccatcgcgc accaggactg gctgaggggc aaggagttca agtgcaaagt ccacaacaag 1020
gcactcccgg cccccatcga gaaaaccatc tccaaagcca gagggcagcc cctggagccg 1080
aaggtctaca ccatgggccc tccccgggag gagctgagca gcaggtcggt cagcctgacc 1140
tgcatgatca acggcttcta cccttccgac atctcggtgg agtgggagaa gaacgggaag 1200
gcagaggaca actacaagac cacgccgacc gtgctggaca gcgacggctc ctacttcctc 1260
tacagcaagc tctcagtgcc cacgagtgag tggcagcggg gcgacgtctt cacctgctcc 1320
gtgatgcacg aggccttgca caaccactac acgcagaagt ccatctcccg ctctccgggt 1380
aaatag 1386
<210> 3
<211> 239
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15
Leu Pro Gly Ala Thr Phe Ala Gln Val Leu Thr Gln Thr Pro Ser Ser
20 25 30
Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser
35 40 45
Gln Thr Leu Tyr Asn Asn Lys Asn Leu Ala Trp Tyr Gln Gln Lys Pro
50 55 60
Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Thr Ser Ser Leu Ala Ser
65 70 75 80
Gly Val Pro Ser Arg Phe Arg Gly Ser Gly Ser Gly Thr Gln Phe Thr
85 90 95
Leu Thr Ile Ser Asp Leu Glu Cys Asp Asp Ala Ala Ala Tyr Tyr Cys
100 105 110
Gln Gly Glu Phe Ser Cys Gly Ser Ala Asp Cys Phe Ala Phe Gly Gly
115 120 125
Gly Thr Glu Val Val Val Lys Gly Asp Pro Val Ala Pro Thr Val Leu
130 135 140
Ile Phe Pro Pro Ala Ala Asp Gln Val Ala Thr Gly Thr Val Thr Ile
145 150 155 160
Val Cys Val Ala Asn Lys Tyr Phe Pro Asp Val Thr Val Thr Trp Glu
165 170 175
Val Asp Gly Thr Thr Gln Thr Thr Gly Ile Glu Asn Ser Lys Thr Pro
180 185 190
Gln Asn Ser Ala Asp Cys Thr Tyr Asn Leu Ser Ser Thr Leu Thr Leu
195 200 205
Thr Ser Thr Gln Tyr Asn Ser His Lys Glu Tyr Thr Cys Lys Val Thr
210 215 220
Gln Gly Thr Thr Ser Val Val Gln Ser Phe Asn Arg Gly Asp Cys
225 230 235
<210> 4
<211> 720
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60
acatttgccc aagtgctgac ccagactcca tcctccgtgt ctgcagctgt gggaggcaca 120
gtcaccatca actgccaggc cagtcagact ctttataata acaaaaattt agcctggtat 180
cagcagaaac cagggcagcc tcccaagctc ctgatctatg gtacatccag tctggcatct 240
ggggtcccat cgcggttcag aggcagtgga tctgggacac aattcactct caccatcagc 300
gacctggagt gtgacgatgc tgccgcttat tattgtcaag gcgaatttag ttgtggtagt 360
gctgattgtt ttgctttcgg cggagggacc gaggtggtgg tcaaaggtga tccagttgca 420
cctactgtcc tcatcttccc accagctgct gatcaggtgg caactggaac agtcaccatc 480
gtgtgtgtgg cgaataaata ctttcccgat gtcaccgtca cctgggaggt ggatggcacc 540
acccaaacaa ctggcatcga gaacagtaaa acaccgcaga attctgcaga ttgtacctac 600
aacctcagca gcactctgac actgaccagc acacagtaca acagccacaa agagtacacc 660
tgcaaggtga cccagggcac gacctcagtc gtccagagct tcaatagggg tgactgttag 720
<210> 5
<211> 461
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly
1 5 10 15
Val Gln Cys Gln Glu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
20 25 30
Pro Glu Gly Ser Leu Thr Leu Thr Cys Lys Ala Ser Gly Phe Asp Phe
35 40 45
Ser Ser Asn Val Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60
Glu Trp Ile Ala Cys Ile Gly Ala Gly Ser Gly Gly Asp Thr Tyr Cys
65 70 75 80
Ala Arg Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Pro Thr
85 90 95
Thr Val Thr Leu Gln Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Ser
100 105 110
Tyr Phe Cys Thr Ser Arg Gly Asp Gly Val Asp Pro Tyr Asp Leu Trp
115 120 125
Gly Pro Gly Thr Leu Val Thr Val Ser Ser Gly Gln Pro Lys Ala Pro
130 135 140
Ser Val Phe Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr
145 150 155 160
Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr
165 170 175
Val Thr Trp Asn Ser Gly Thr Leu Thr Asn Gly Val Arg Thr Phe Pro
180 185 190
Ser Val Arg Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Ser
195 200 205
Val Thr Ser Ser Ser Gln Pro Val Thr Cys Asn Val Ala His Pro Ala
210 215 220
Thr Asn Thr Lys Val Asp Lys Thr Val Ala Pro Ser Thr Cys Ser Lys
225 230 235 240
Pro Met Cys Pro Pro Pro Glu Leu Pro Gly Gly Pro Ser Val Phe Ile
245 250 255
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
260 265 270
Val Thr Cys Val Val Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln
275 280 285
Phe Thr Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro
290 295 300
Leu Arg Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu
305 310 315 320
Pro Ile Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys
325 330 335
Val His Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
340 345 350
Ala Arg Gly Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro
355 360 365
Arg Glu Glu Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn
370 375 380
Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys
385 390 395 400
Ala Glu Asp Asn Tyr Lys Thr Thr Pro Thr Val Leu Asp Ser Asp Gly
405 410 415
Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln
420 425 430
Arg Gly Asp Val Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn
435 440 445
His Tyr Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys
450 455 460
<210> 6
<211> 1386
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60
gagcagctgg tggagtccgg gggaggcctg gtccagcctg agggatccct gacactcacc 120
tgcaaagcct ctggattcga cttcagtagc aatgtaatgt gctgggtccg ccaggctcca 180
gggaagggac tggaatggat cgcatgcatt ggcgctggta gtggtggtga tacttactgc 240
gcgaggtggg cgaagggccg attcaccatc tccaaaacct cgccgaccac ggtgactctg 300
caaatgacca gtctgacagc cgcggacacg gcctcctatt tctgtacgag taggggtgat 360
ggtgttgatc cttatgactt gtggggccca ggcaccctgg tcaccgtctc ctcagggcaa 420
cctaaggctc catcagtctt cccactggcc ccctgctgcg gggacacacc cagctccacg 480
gtgaccctgg gctgcctggt caaaggctac ctcccggagc cagtgaccgt gacctggaac 540
tcgggcaccc tcaccaatgg ggtacgcacc ttcccgtccg tccggcagtc ctcaggcctc 600
tactcgctga gcagcgtggt gagcgtgacc tcaagcagcc agcccgtcac ctgcaacgtg 660
gcccacccag ccaccaacac caaagtggac aagaccgttg cgccctcgac atgcagcaag 720
cccatgtgcc caccccctga actcccgggg ggaccgtctg tcttcatctt ccccccaaaa 780
cccaaggaca ccctcatgat ctcacgcacc cccgaggtca catgcgtggt ggtggacgtg 840
agccaggatg accccgaggt gcagttcaca tggtacataa acaacgagca ggtgcgcacc 900
gcccggccgc cgctacggga gcagcagttc aacagcacga tccgcgtggt cagcaccctc 960
cccatcgcgc accaggactg gctgaggggc aaggagttca agtgcaaagt ccacaacaag 1020
gcactcccgg cccccatcga gaaaaccatc tccaaagcca gagggcagcc cctggagccg 1080
aaggtctaca ccatgggccc tccccgggag gagctgagca gcaggtcggt cagcctgacc 1140
tgcatgatca acggcttcta cccttccgac atctcggtgg agtgggagaa gaacgggaag 1200
gcagaggaca actacaagac cacgccgacc gtgctggaca gcgacggctc ctacttcctc 1260
tacagcaagc tctcagtgcc cacgagtgag tggcagcggg gcgacgtctt cacctgctcc 1320
gtgatgcacg aggccttgca caaccactac acgcagaagt ccatctcccg ctctccgggt 1380
aaatag 1386
<210> 7
<211> 239
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15
Leu Pro Gly Ala Thr Phe Ala Gln Val Leu Thr Gln Thr Ala Ser Pro
20 25 30
Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser
35 40 45
Gln Ser Val Tyr Ser Asn Asn Tyr Leu Ser Trp Phe Gln Gln Lys Pro
50 55 60
Gly Gln Pro Pro Lys Gln Leu Ile Tyr Asp Ala Ser Thr Leu Ala Ser
65 70 75 80
Gly Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr
85 90 95
Leu Thr Ile Thr Asp Val Leu Cys Asp Asp Ala Ala Thr Tyr Tyr Cys
100 105 110
Leu Gly Gly Tyr Asp Cys Ser Ser Ala Asp Cys Trp Ala Phe Gly Gly
115 120 125
Gly Thr Glu Val Val Val Lys Gly Asp Pro Val Ala Pro Thr Val Leu
130 135 140
Ile Phe Pro Pro Ala Ala Asp Gln Val Ala Thr Gly Thr Val Thr Ile
145 150 155 160
Val Cys Val Ala Asn Lys Tyr Phe Pro Asp Val Thr Val Thr Trp Glu
165 170 175
Val Asp Gly Thr Thr Gln Thr Thr Gly Ile Glu Asn Ser Lys Thr Pro
180 185 190
Gln Asn Ser Ala Asp Cys Thr Tyr Asn Leu Ser Ser Thr Leu Thr Leu
195 200 205
Thr Ser Thr Gln Tyr Asn Ser His Lys Glu Tyr Thr Cys Lys Val Thr
210 215 220
Gln Gly Thr Thr Ser Val Val Gln Ser Phe Asn Arg Gly Asp Cys
225 230 235
<210> 8
<211> 720
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60
acatttgccc aagtgctgac ccagactgca tcccccgtgt ctgcagctgt gggaggcaca 120
gtcaccatca actgccaggc cagtcagagt gtttatagta acaattactt atcctggttt 180
cagcagaaac cagggcagcc tcccaagcaa ctgatctatg atgcatccac tctggcatct 240
ggggtcccat cgcggttcaa aggcagtgga tctgggacac agttcactct caccatcacc 300
gacgtactgt gtgacgatgc tgccacttac tactgtctag gcggttatga ttgtagtagt 360
gctgattgtt gggctttcgg cggagggacc gaggtggtgg tcaaaggtga tccagttgca 420
cctactgtcc tcatcttccc accagctgct gatcaggtgg caactggaac agtcaccatc 480
gtgtgtgtgg cgaataaata ctttcccgat gtcaccgtca cctgggaggt ggatggcacc 540
acccaaacaa ctggcatcga gaacagtaaa acaccgcaga attctgcaga ttgtacctac 600
aacctcagca gcactctgac actgaccagc acacagtaca acagccacaa agagtacacc 660
tgcaaggtga cccagggcac gacctcagtc gtccagagct tcaatagggg tgactgttag 720
<210> 9
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ccgtccaagc ttatggagac tgggctgcgc tggc 34
<210> 10
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
caacaaggat ccctatttac ccggagagcg ggag 34
<210> 11
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ccgtccaagc ttatggacac gagggccccc actc 34
<210> 12
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
caacaaggat ccctaacagt cacccctatt gaagc 35

Claims (10)

1.一种抗IP-10单克隆抗体，其特征在于，包括重链可变区和轻链可变区；
所述重链可变区的氨基酸序列如SEQ ID NO:1所示；
所述轻链可变区的氨基酸序列如SEQ ID NO:3所示。
2.一种抗IP-10单克隆抗体的编码DNA，其特征在于，编码如权利要求1所述的抗IP-10单克隆抗体，其DNA序列包括重链可变区和轻链可变区的编码DNA序列；
所述重链可变区的编码DNA序列如SEQ ID NO:2所示；
所述轻链可变区的编码DNA序列如SEQ ID NO:4所示。
3.一种抗IP-10单克隆抗体，其特征在于，包括重链可变区和轻链可变区；
所述重链可变区的氨基酸序列如SEQ ID NO:5所示；
所述轻链可变区的氨基酸序列如SEQ ID NO:7所示。
4.一种抗IP-10单克隆抗体的编码DNA，其特征在于，编码如权利要求3所述的抗IP-10单克隆抗体，其DNA序列包括重链可变区和轻链可变区的编码DNA序列；
所述重链可变区的编码DNA序列如SEQ ID NO:6所示；
所述轻链可变区的编码DNA序列如SEQ ID NO:8所示。
5.一种抗IP-10单克隆抗体的制备方法，其特征在于，如权利要求1或3所述的抗IP-10单克隆抗体，其制备方法包括以下步骤：
1)用人IP-10胞外免疫兔，作出免疫反应后，处死，取脾脏，分离获得脾脏细胞；
2)筛选获得能特异性结合人IP-10的B细胞；
3)将B细胞进行亚克隆，获得抗体重链和轻链的可变区编码序列；
4)获得的可变区编码序列进行重组、转染、纯化后获得抗IP-10单克隆抗体。
6.药物组合物，其特征在于，包括如权利要求1或3所述的抗IP-10单克隆抗体和药学上可接受的载体。
7.一种表达载体，其特征在于，包含如权利要求2或4所述的抗IP-10单克隆抗体的编码DNA，分别用于表达如权利要求1或3所述的抗IP-10单克隆抗体。
8.一种原核或真核宿主细胞，其特征在于，包含如权利要求7所述的表达载体。
9.一种如权利要求1或3所述的抗IP-10单克隆抗体在制备治疗或预防人病毒感染、肿瘤及炎性疾病药物中的用途。
10.一种用于检测IP-10抗原或sIP-10抗原的试剂盒，其特征在于，包括如权利要求1或3所述的抗IP-10单克隆抗体。
CN202210211321.0A 2022-03-05 2022-03-05 一种抗ip-10单克隆抗体 Active CN114656561B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210211321.0A CN114656561B (zh) 2022-03-05 2022-03-05 一种抗ip-10单克隆抗体

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210211321.0A CN114656561B (zh) 2022-03-05 2022-03-05 一种抗ip-10单克隆抗体

Publications (2)

Publication Number Publication Date
CN114656561A true CN114656561A (zh) 2022-06-24
CN114656561B CN114656561B (zh) 2023-06-09

Family

ID=82027896

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210211321.0A Active CN114656561B (zh) 2022-03-05 2022-03-05 一种抗ip-10单克隆抗体

Country Status (1)

Country Link
CN (1) CN114656561B (zh)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005060457A2 (en) * 2003-12-04 2005-07-07 Pdl Biopharma, Inc. Treatment of inflammatory bowel diseases with anti-ip-10 antibodies
CA2478138A1 (en) * 2004-08-17 2006-02-17 University Health Network Cxcl10-based diagnosis and treatment of respiratory illnesses
CN1889979A (zh) * 2003-12-10 2007-01-03 米德列斯公司 Ip－10抗体及其用途
JP2008031143A (ja) * 2006-07-26 2008-02-14 Chemokine Therapeutics Corp ヒト疾患を治療するためのインターフェロン誘導性タンパク質−１０（ｉｐ−１０又はｃｘｃｌ１０）ケモカイン類縁体の設計
US20080063646A1 (en) * 2003-12-04 2008-03-13 Balaji Balasa Treatment Of Inflammatory Bowel Diseases With Anti-Ip-10 Antibodies
US20090169561A1 (en) * 2007-02-28 2009-07-02 Novimmune S.A. Anti-IP-10 antibodies and methods of use thereof
AU2011250741A1 (en) * 2003-12-10 2011-12-08 E. R. Squibb & Sons, L.L.C. IP-10 antibodies and their uses

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005060457A2 (en) * 2003-12-04 2005-07-07 Pdl Biopharma, Inc. Treatment of inflammatory bowel diseases with anti-ip-10 antibodies
US20080063646A1 (en) * 2003-12-04 2008-03-13 Balaji Balasa Treatment Of Inflammatory Bowel Diseases With Anti-Ip-10 Antibodies
CN1889979A (zh) * 2003-12-10 2007-01-03 米德列斯公司 Ip－10抗体及其用途
AU2011250741A1 (en) * 2003-12-10 2011-12-08 E. R. Squibb & Sons, L.L.C. IP-10 antibodies and their uses
CA2478138A1 (en) * 2004-08-17 2006-02-17 University Health Network Cxcl10-based diagnosis and treatment of respiratory illnesses
JP2008031143A (ja) * 2006-07-26 2008-02-14 Chemokine Therapeutics Corp ヒト疾患を治療するためのインターフェロン誘導性タンパク質−１０（ｉｐ−１０又はｃｘｃｌ１０）ケモカイン類縁体の設計
US20090169561A1 (en) * 2007-02-28 2009-07-02 Novimmune S.A. Anti-IP-10 antibodies and methods of use thereof

Also Published As

Publication number Publication date
CN114656561B (zh) 2023-06-09

Similar Documents

Publication Publication Date Title
CN110684740B (zh) 一种抗人泛素羧基末端水解酶-1(uch-l1)的单克隆抗体及其应用
US10494430B2 (en) Anti-active GIP antibody
CN112920275B (zh) 可特异性结合sST2的结合蛋白、试剂和试剂盒
JP2021181450A (ja) 細胞内抗原に対して向けられた単一ドメイン抗体
CN113045646B (zh) 抗新型冠状病毒SARS-CoV-2的抗体
US20120083009A1 (en) Rheumatoid Arthritis Test Method And Treating Method
CN114656561B (zh) 一种抗ip-10单克隆抗体
CN110386973B (zh) 具有多种功能的蛋白多肽及其医药用途
CN115925927A (zh) 一种抗il-10单克隆抗体及其制备方法和应用
CN111363042B (zh) 一种高特异性抗小鼠cd226单克隆抗体及其应用
CN112574303B (zh) 一种抗c反应蛋白的抗体
KR20070042994A (ko) 항시노비올린 항체
CN110894236B (zh) 一种抗曲霉菌半乳甘露聚糖的单克隆抗体及其应用
WO2019230874A1 (ja) ヒト由来サンプルにおける可溶型ｔｌｒ７の分析
KR20120113580A (ko) 비브리오 패혈증균 알티엑스에이원에 특이적인 단일클론항체, 이를 분비하는 하이브리도마 및 이를 포함하는 진단키트
JPH0767689A (ja) 抗ｌｄ７８ポリペプチドモノクローン抗体
CN118146365B (zh) 一种抗bp230高亲和力单克隆抗体及其应用
CN117327177B (zh) 抗脂联素抗体、检测脂联素的试剂和试剂盒
CN112724251B (zh) 一种包含c反应蛋白抗原结合结构域的结合蛋白
CN114933652B (zh) 一种抗念珠菌甘露聚糖单克隆抗体及其应用
CN112500486B (zh) 一种抗pd-l1抗体及其应用
JP4381145B2 (ja) 細胞外グラニュライシンの検出方法
US7893199B2 (en) Peptides having neutrophil-stimulating activity
KR20040068559A (ko) 심장 손상으로부터 심장을 보호하기 위한트리스테트라프롤린의 저해
CN118126174A (zh) 一种抗bp230高亲和力单克隆抗体及其应用

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