CN106866823A - 一种抗Her2的纳米抗体 - Google Patents

一种抗Her2的纳米抗体 Download PDF

Info

Publication number
CN106866823A
CN106866823A CN201710136524.7A CN201710136524A CN106866823A CN 106866823 A CN106866823 A CN 106866823A CN 201710136524 A CN201710136524 A CN 201710136524A CN 106866823 A CN106866823 A CN 106866823A
Authority
CN
China
Prior art keywords
gly
ser
ala
thr
val
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.)
Pending
Application number
CN201710136524.7A
Other languages
English (en)
Inventor
李洪利
凌志
庄文超
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.)
Health (beijing) Biotechnology Co Ltd
Original Assignee
Health (beijing) Biotechnology Co Ltd
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 Health (beijing) Biotechnology Co Ltd filed Critical Health (beijing) Biotechnology Co Ltd
Priority to CN201710136524.7A priority Critical patent/CN106866823A/zh
Publication of CN106866823A publication Critical patent/CN106866823A/zh
Pending legal-status Critical Current

Links

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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • 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
    • G01N33/6854—Immunoglobulins
    • 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
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • 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
    • C12N2800/101—Plasmid DNA for bacteria
    • 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
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators

Landscapes

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

Abstract

本发明公开了一种抗Her2的纳米抗体，所述抗体的氨基酸编码序列：SEQ ID NO：8到SEQ ID NO：14，编码上述氨基酸序列的DNA序列：SEQ ID NO：1至SEQ ID NO：7。

Description

一种抗Her2的纳米抗体
技术领域
本发明属于生物医学或生物制药技术领域，涉及一种针对于Her2的纳米抗体。
背景技术
人表皮生长因子受体-2(Her2)是迄今为止乳腺癌中研究较为透彻的基因之一，于20世纪80年代分别由三个研究小组独立发现。Her2基因的过表达不仅与肿瘤的发生发展相关外，还是一个重要的临床治疗监测及预后指标，并且是肿瘤靶向治疗药物选择的一个重要靶点。血清Her2与组织学Her2、乳腺癌患者的肿瘤负荷、***状况均有一定关联，并可能对化疗或内分泌治疗疗效产生一定影响，可能是一个独立的预后因素。
人类表皮生长因子受体(human epidermal growth factor receptor)家族有4个成员：EGFR、Her2(人类表皮生长因子受体2)、ErbB3和ErbB4，广泛表达于上皮、间质及神经组织，生理状态下它们参与激活一系列复杂的细胞信号转导途径，调控正常组织细胞的生长、***、分化等重要生理过程。然而研究表明某些肿瘤的发生、发展及恶性生物学行为与EGFR家族的异常表达和活化关系密切(Albanell J,CodonyJ,Rovira A,Mellado B,GascónP.Mechanism of action of anti-Her2monoclonal antibodies:scientific update ontrastuzumab and2C4.Adv Exp Med Biol.2003；532:253-68)。Her2没有天然配体，在单体状态下无活性，当形成二聚体时可使酪氨酸激酶活化，激活MAPK和PI3K/Akt通路，最终导致肿瘤细胞增殖和存活(Hudis CA.Trastuzumab-mechanism of action and use inclinical practice.N Engl J Med.2007Jul5；357(1):39-51)。
1993年比利时科学家首次在Nature报道：在骆驼血液中的抗体，有一半没有轻链，而且更让人惊喜的是，这些缺失轻链的“重链抗体”能像正常抗体一样与抗原等靶标紧密结合，另外不像scFv(人工改造的单链抗体片段)那样互相粘连，甚至聚集成块。这种抗体只包含一个重链可变区和两个常规的CH2与CH3区，更重要的是单独克隆并表达出来的VHH区(重链可变区)具有很好的结构稳定性与抗原结合活性，分子量只是普通抗体的1/10，所以VHH也称Nanobody(纳米抗体)；与此同时纳米抗体化学性质也更加灵活，稳定性好，可溶性高，表达容易且容易获得，容易偶联其他分子，因此应用纳米抗体技术研发Her2检测试剂具有广阔的前景。
发明内容
发明目的：本发明所要解决的技术问题是提供一种针对Her2表位的纳米抗体，同时提供该纳米抗体的编码的氨基酸序列以及核苷酸序列。
技术方案：为实现上述目的，本发明的第一方面，提供了一种Her2的纳米抗体的VHH链，选自下组的FR的氨基酸序列：SEQ ID NO：1所示的VHH1，SEQ ID NO：2所示的VHH2，SEQ ID NO：3所示的VHH3，SEQ ID NO：4所示的VHH4；或SEQ ID NO：5所示的VHH5，SEQ IDNO：6所示的VHH6，SEQ ID NO：7所示的VHH7。
本发明第2方面，提供了一种DNA分子，它编码选自下组的蛋白质：本发明所述的Her2的纳米抗体的VHH链，或本发明所述的Her2纳米抗体。优选地，所述的DNA分子，其特征在于，它具有选自下组的DNA序列：SEQ ID NO：8至SEQ ID NO：14
本发明的第3方面，提供了一种表达载体，它含SEQ ID NO：8至SEQ ID NO：14所示的核苷酸序列。
本发明的第4方面，提供了本发明所述的Her2纳米抗体用于检测Her2的用途。
有益效果：与现有技术相比，本发明的优点如下：本发明将Her2抗原免疫羊驼，随后利用该骆驼外周血淋巴细胞进行流式细胞筛选，得到阳性结果之后提取RNA，进行反转录获得cDNA。从而获得了针对Her2特异性的纳米抗体基因，将此基因转至大肠杆菌中，从而建立了能在大肠杆菌中高效表达的纳米抗体株。
附图说明
图1是纳米抗体的基因电泳图；其中泳道1是DL2000DNA分子标准，DL2000(2000bp,1000bp,750bp,500bp,250bp,100bp)泳道2-8是PCR扩增重链抗体可变区片段
图2是表达的Her2纳米抗体，经镍柱树脂凝胶亲和层析纯化后的SDS-PAGE的电泳图；其中泳道5是蛋白分子标准，泳道1-4以及6-8是破菌后蛋白总的粗提液样品。蛋白分子标准是：
116.0KD,66.2KD,45.0KD,35.0KD,25.0KD,18.4KD,14.4KD.目的蛋白约为20KD。
具体实施方式
本发明将Her2抗原免疫羊驼，随后利用该骆驼外周血淋巴细胞进行流式细胞筛选，得到阳性结果之后提取RNA，进行反转录获得cDNA。从而获得了针对Her2特异性的纳米抗体基因，将此基因转至大肠杆菌中，从而建立了能在大肠杆菌中高效表达的纳米抗体株。
下面结合具体实施例，进一步阐述本发明。
实施例1：针对于Her2的纳米抗体文库的构建：
(1)将购自ACROBiosystems公司的Her2抗原，其序列为GenBank:aaa75493，浓度为100微克每毫升，每次免疫将100微克Her2与弗氏佐剂等体积混合，免疫一只羊驼，每周一次，共免疫4次，除第一次使用完全的弗氏佐剂，剩余几次全部使用弗式不全佐剂，免疫过程中刺激B细胞表达抗原特异性的纳米抗体。
(2)4次免疫结束后，分离外周血单核细胞，流式分选阳性细胞并提取总RNA，参照QIAGEN公司提供的RNA提取试剂盒。(3)按照Super-Script III FIRST STRANDSUPERMIX试剂盒说明书，将提取的RNA反转录成cDNA并利用套式PCR扩增VHH链，
第一轮PCR：
上游引物：CAGGTGAAGGTCATCGARTC
下游:GATGCTCTTGTGACTCAGGAATC
扩增获得羊驼单链和双链的重链可变区-恒定区(VH-CH)，53℃退火,35个循环；
第二轮PCR：
以第一轮PCR产物作模板，
上游引物：
GGAATTCCATATGGATTATAAAGATGATGATAAACGCAGAGACAGTGACCAGAGT
下游引物：CCACGATTCTGCGGCCGCTTAATCCAGCTGACTCAGCC
扩增获得羊驼单链VHH,53℃退火,35个循环,回收目的片段，结果如图1显示。从左到右的DNA条带分别是：第一为DL2000的分子Marker，第2-8泳带为目的基因。
实施例2：纳米抗体在宿主菌大肠杆菌中表达、纯化：
1.构建表达载体
将合成的纳米抗体DNA片段双酶切连入表达载体pET28a中，测序正确后在表达宿主中表达。表达载体的酶切条件为载体(1ug)35μL，10倍浓度快切酶缓冲液10μL，两种酶NdeI、NotI各2μL，水51μL，37℃水浴20min，80℃失活10min，琼脂糖凝胶电泳回收定量。DNA片段酶切条件为DNA片段1ug 20μL，10倍浓度快切酶缓冲液10μL，两种酶NdeI、NotI各2μL，水66μL，37℃水浴20min，80℃失活10min，琼脂糖凝胶电泳回收定量。表达载体与DNA片段连接条件：表达载体(50ng/μL)1μL，DNA片段(40ng/μL)1.5μL，10倍浓度的T4脱氧核糖核酸缓冲液1μL，T4脱氧核糖核酸连接酶1μL，水5.5μL，室温1h连接，转入BL21大肠杆菌感受态。
2.诱导表达
将序列正确的样品进行诱导表达，诱导表达步骤为：将正确序列样本菌液与2YT培养基按1:100稀释，37℃，180～200rpm进行摇菌，摇至菌液OD600值为0.6～1.0时，取出少量菌液，加入终浓度为1mmol/L的IPTG进行诱导表达3～4h，收集菌液，空载体pet28a菌液按照上述正确样本菌液相同诱导表达步骤操作；以未诱导的空载体pet28a菌液、诱导的空载体pet28a菌液和未诱导的正确样本菌液作为对照进行聚丙烯酰胺凝胶电泳试验，结果表明IPTG能够诱导抗CD3e纳米抗体在pet28a菌液中的表达。
3.纯化蛋白
将IPTG诱导、表达后的菌液于离心机中7000rpm，离心10min收集菌体，用缓冲液PBS(PH7.4)清洗一次，再次离心收集菌体；用10mL的PBS(PH7.4)将菌液吹打混匀，置于冰上，放到超声波细胞粉碎仪中，按超声发振时间5s，间隙时间5s，保护温度60℃，超声功率10％的程序超声破碎10min，将破碎后的菌液放于离心机中7000rpm，离心10min，将上清液进行亲和层析纯化，保存备用。
实施例3 Her2抗体酶联免疫活性检测
Her2抗原用包被液PH(9.6)稀释到2ng/μL，按每孔100μL加入到酶标板中，放置于4℃冰箱中过夜，第二天进行ELISA实验，ELISA步骤如下：
步骤1、按每孔200μL的PBS-T加入包被后的酶标板进行清洗，清洗5次，每次3min，甩掉清洗液，于纸巾上拍板确保洗液全部被吸干；
步骤2、按每孔200μL向酶标板中加入5％的脱脂奶粉，室温封闭1h；
步骤3、按步骤1中清洗步骤进行清洗，然后，依次将100μL的空白对照液、阴性对照、纯化后的Her2抗体加入到酶标板中，室温孵育2h；
步骤4、按步骤1中清洗步骤进行清洗，然后，按每孔100μL向酶标板加入HRP标记的标签抗体，室温孵育1h；
步骤5按步骤1中清洗步骤进行清洗，然后向每孔加入100μL的TMB，避光放置10min；
步骤6、向每孔加入50μL的2mol/L的硫酸溶液，终止反应，将酶标板放于酶标仪中，于450nm条件下读数。
结果如下：
其中，阴性对照为：未连接抗体基因的空载体培养液
空白对照为：PBS洗液
人乳腺癌细胞系SK-br-3细胞购自国家细胞资源共享平台
最后应说明的是：显然，上述实施例仅仅是为清楚地说明本申请所作的举例，是优选的实施例。而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本申请型的保护范围之中。
SEQUENCE LISTING
<110> 康众（北京）生物科技有限公司
<120> 一种抗HER2的纳米抗体
<130> 0
<160> 14
<170> PatentIn version 3.3
<210> 1
<211> 405
<212> DNA
<213> Vicugna pacos
<400> 1
atgggtcagg tgcagctggt ggagtctggg ggaggcttgg tgcaggctgg ggactctctg 60
agactctcct gagcagcctc gggacccagt tttagtgact atgccgtggg ctggttccgc 120
caggctatag ggaaggagcg tgaatttgta ggcgctgtta gctggacgag cacagaaaca 180
tcctatgcgg acgctgtgaa ggggcgattc accatctcca gagacaacgc caagaacacg 240
gtgtatctgc aaatgaacag cctgaagcct gaggacacgg ccgtttatac ttgtgcagca 300
actagaacgt acggctttac ttcacgttat caaactaact atgagtactg gggccagggg 360
acccaggtca ccgtctcctc agaacccaag acaccaaaac cacaa 405
<210> 2
<211> 405
<212> DNA
<213> Vicugna pacos
<400> 2
atgggtcagg tgcagctcgt ggagtctggg ggaggcttgg tgcaggctgg ggactctctg 60
agactctcct gagcagcctc gggacccagt tttagtgact atgccgtggg ctggttccgc 120
caggctatag ggaaggagcg tgaatttgta ggcgctgtta gctggacgag cacagaaaca 180
tcctatgcgg acgctgtgaa ggggcgattc accatctcca gagacaacgc caagaacacg 240
gtgtatctgc aaatgaacag cctgaagcct gaggacacgg ccgtttatac ttgtgcagca 300
actagaacgt acggctttac ttcacgttat caaactaact atgagtactg gggccagggg 360
acccaggtca ccgtctcctc agaacccaag acaccaaaac cacaa 405
<210> 3
<211> 402
<212> DNA
<213> Vicugna pacos
<400> 3
atgggtcagg tgcagctcgt ggagtctggg ggaggcttgg tgcagcctgg ggggtctctg 60
agactctcct gtgcagcctc tggacgcacc ttcagtagct atcgaatggg ctggttccgc 120
caggctccag ggaaggagcg tgagtttgta gcagctatta gctggtgtgg tggtgtcaca 180
ttctatgcaa actccgtgaa gggccgattc accatctcca gagacaacgc caagaacacg 240
gtgtatctgc aaatgaacag cctgaaacca gaggacacgg ccgtctatta ctgtaatgtg 300
aagggcctta cgaacgtagc tggaacgtgg gtttcgggtg actactgggg ccaggggacc 360
caggtcaccg tctcctcagc gcaccacagc gaagacccca gc 402
<210> 4
<211> 399
<212> DNA
<213> Vicugna pacos
<400> 4
atgggtcagg tgcagctcgt ggagtctggg ggaggcttgg tgcagcctgg ggggtctctg 60
agactctcct gagcagcctc tggaagcatc agcaccgtcg atatcatggg ctggtaccgc 120
caggctgtag ggaagcagcg cgagttggtc gcggttgtta ctaggagtgc tggcgcccgc 180
tatgcagact ccgtgaaggg ccgattcacc atctccagag acaatgtcaa gagcacggtc 240
tatctgcaaa tgaacagcct gaaacctgag gacacggccg tttattactg tgcactgact 300
aatcgcggct atgacctcgg gcggccggcg gagtatccct actggggcca ggggacccag 360
gtcaccgtcc cctcagaacc caagacacca aaaccacaa 399
<210> 5
<211> 405
<212> DNA
<213> Vicugna pacos
<400> 5
atgggtcagg tgcagctcgt ggagtctggg ggaggattgg tgcaggctgg gggctctctg 60
agactctcct gtgcagcctc tggacgcacc ttcagtgcct atgacatggg ctggttccgc 120
caggctccag ggaaggagcg tgagtttgta gcagctatta gttggagtgg cggtaggaca 180
tattatggag actccgtgaa gggccgattc accatctcca gagacaacgc caagaacctg 240
gtatatctgc aaatgaacag cctgaaacct gaggacacgg ccgtttatta ctgtgcagct 300
gggcggaata tatattttag taatagtccc ctggcggggt cggactactg gggccagggg 360
acccaggtca ccgtctcctc agaacccaag acaccaaaac cacaa 405
<210> 6
<211> 405
<212> DNA
<213> Vicugna pacos
<400> 6
atgggtcagg tgcagctcgt ggagtctggg ggaggattgg tgcaggctgg gggctctctg 60
agactctcct gtgcagcctc tggacgcacc ttcagtgcct atgacatggg ctggttccgc 120
caggctccag ggaaggagcg tgagtttgta gcagctatta gttggagtgg cggtaggaca 180
tattatggag actccgtgaa gggccgattc accatctcca gagacaacgc caagaacctg 240
gtatatctgc aaatgaacag cctgaaacct gaggacacgg ccgtttatta ctgtgcagct 300
gggcggaata tatattttag taatagtccc ctggcggggt cggactactg gggccagggg 360
acccaggtca ccgtctcctc agaacccaag acaccaaaac cacaa 405
<210> 7
<211> 405
<212> DNA
<213> Vicugna pacos
<400> 7
atgggtcagg tgcagctcgt ggagtctggg ggaggcttgg tgcaggctgg ggactctctg 60
agactctcct gtgcagcctc gggacccagt tttagtgact atgccgtggg ctggttccgc 120
caggctatag ggaaggagcg tgaatttgta ggcgctgtta gctggacgag cacagaaaca 180
tcctatgcgg acgctgtgaa ggggcgattc accatctcca gagacaacgc caagaacacg 240
gtgtatctgc aaatgaacag cctgaagcct gaggacacgg ccgtttatac ttgtgcagca 300
actagaacgt acggctttac ttcacgttat caaactaact atgagtactg gggccagggg 360
acccaggtca ccgtctcctc agaacccaag acaccaaaac cacaa 405
<210> 8
<211> 134
<212> PRT
<213> Vicugna pacos
<400> 8
Met Gly Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
1 5 10 15
Gly Asp Ser Leu Arg Leu Ser Ala Ala Ser Gly Pro Ser Phe Ser Asp
20 25 30
Tyr Ala Val Gly Trp Phe Arg Gln Ala Ile Gly Lys Glu Arg Glu Phe
35 40 45
Val Gly Ala Val Ser Trp Thr Ser Thr Glu Thr Ser Tyr Ala Asp Ala
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Thr
85 90 95
Cys Ala Ala Thr Arg Thr Tyr Gly Phe Thr Ser Arg Tyr Gln Thr Asn
100 105 110
Tyr Glu Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro
115 120 125
Lys Thr Pro Lys Pro Gln
130
<210> 9
<211> 134
<212> PRT
<213> Vicugna pacos
<400> 9
Met Gly Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
1 5 10 15
Gly Asp Ser Leu Arg Leu Ser Ala Ala Ser Gly Pro Ser Phe Ser Asp
20 25 30
Tyr Ala Val Gly Trp Phe Arg Gln Ala Ile Gly Lys Glu Arg Glu Phe
35 40 45
Val Gly Ala Val Ser Trp Thr Ser Thr Glu Thr Ser Tyr Ala Asp Ala
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Thr
85 90 95
Cys Ala Ala Thr Arg Thr Tyr Gly Phe Thr Ser Arg Tyr Gln Thr Asn
100 105 110
Tyr Glu Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro
115 120 125
Lys Thr Pro Lys Pro Gln
130
<210> 10
<211> 134
<212> PRT
<213> Vicugna pacos
<400> 10
Met Gly Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
1 5 10 15
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser
20 25 30
Ser Tyr Arg Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Phe Val Ala Ala Ile Ser Trp Cys Gly Gly Val Thr Phe Tyr Ala Asn
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
65 70 75 80
Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Asn Val Lys Gly Leu Thr Asn Val Ala Gly Thr Trp Val Ser
100 105 110
Gly Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala His
115 120 125
His Ser Glu Asp Pro Ser
130
<210> 11
<211> 132
<212> PRT
<213> Vicugna pacos
<400> 11
Met Gly Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
1 5 10 15
Gly Gly Ser Leu Arg Leu Ser Ala Ala Ser Gly Ser Ile Ser Thr Val
20 25 30
Asp Ile Met Gly Trp Tyr Arg Gln Ala Val Gly Lys Gln Arg Glu Leu
35 40 45
Val Ala Val Val Thr Arg Ser Ala Gly Ala Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Ser Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Leu Thr Asn Arg Gly Tyr Asp Leu Gly Arg Pro Ala Glu Tyr Pro
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Pro Ser Glu Pro Lys Thr
115 120 125
Pro Lys Pro Gln
130
<210> 12
<211> 135
<212> PRT
<213> Vicugna pacos
<400> 12
Met Gly Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
1 5 10 15
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser
20 25 30
Ala Tyr Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Phe Val Ala Ala Ile Ser Trp Ser Gly Gly Arg Thr Tyr Tyr Gly Asp
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Leu
65 70 75 80
Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Ala Gly Arg Asn Ile Tyr Phe Ser Asn Ser Pro Leu Ala
100 105 110
Gly Ser Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu
115 120 125
Pro Lys Thr Pro Lys Pro Gln
130 135
<210> 13
<211> 135
<212> PRT
<213> VICUGNA PACOS
<400> 13
Met Gly Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
1 5 10 15
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser
20 25 30
Ala Tyr Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
35 40 45
Phe Val Ala Ala Ile Ser Trp Ser Gly Gly Arg Thr Tyr Tyr Gly Asp
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Leu
65 70 75 80
Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Ala Gly Arg Asn Ile Tyr Phe Ser Asn Ser Pro Leu Ala
100 105 110
Gly Ser Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu
115 120 125
Pro Lys Thr Pro Lys Pro Gln
130 135
<210> 14
<211> 135
<212> PRT
<213> Vicugna pacos
<400> 14
Met Gly Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
1 5 10 15
Gly Asp Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Pro Ser Phe Ser
20 25 30
Asp Tyr Ala Val Gly Trp Phe Arg Gln Ala Ile Gly Lys Glu Arg Glu
35 40 45
Phe Val Gly Ala Val Ser Trp Thr Ser Thr Glu Thr Ser Tyr Ala Asp
50 55 60
Ala Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
65 70 75 80
Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
85 90 95
Thr Cys Ala Ala Thr Arg Thr Tyr Gly Phe Thr Ser Arg Tyr Gln Thr
100 105 110
Asn Tyr Glu Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu
115 120 125
Pro Lys Thr Pro Lys Pro Gln
130 135

Claims (3)

1.一种抗Her2的纳米抗体的 VHH 链，其特征在于：所述的 VHH 链的氨基酸序列为SEQ ID NO ：8至SEQ ID NO ：14 所示的氨基酸序列之一。
2.一种Her2纳米抗体，其特征在于: 它针对Her2表位的纳米抗体，所述的纳米抗体的VHH 链的氨基酸序列为权利要求 1 所述的氨基酸序列。
3.一种 DNA 分子，其特征在于：它编码选自下组的蛋白质 ：权利要求 1 所述的 Her2的纳米抗体的 VHH 链。
CN201710136524.7A 2017-03-08 2017-03-08 一种抗Her2的纳米抗体 Pending CN106866823A (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710136524.7A CN106866823A (zh) 2017-03-08 2017-03-08 一种抗Her2的纳米抗体

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710136524.7A CN106866823A (zh) 2017-03-08 2017-03-08 一种抗Her2的纳米抗体

Publications (1)

Publication Number Publication Date
CN106866823A true CN106866823A (zh) 2017-06-20

Family

ID=59170192

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710136524.7A Pending CN106866823A (zh) 2017-03-08 2017-03-08 一种抗Her2的纳米抗体

Country Status (1)

Country Link
CN (1) CN106866823A (zh)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018233624A1 (zh) * 2017-06-20 2018-12-27 和迈生物科技有限公司 抗Her2纳米抗体及其编码序列和用途
CN109627319A (zh) * 2019-01-11 2019-04-16 北京协同创新研究院 一种抗her-2的重链抗体及其应用
CN110950967A (zh) * 2019-12-13 2020-04-03 山东民康生物科技有限公司 抗人血清白蛋白纳米抗体与il-2融合蛋白及制备方法
CN114085287A (zh) * 2021-11-12 2022-02-25 中国农业科学院北京畜牧兽医研究所 犬细小病毒纳米抗体cpv-vhh-f5及其应用
CN114409787A (zh) * 2022-02-18 2022-04-29 南京英瀚斯生物科技有限公司 一种针对her3纳米抗体的分离纯化方法
CN116574186A (zh) * 2023-07-07 2023-08-11 云南大学 特异性结合her2的纳米抗体及其应用
WO2024001844A1 (zh) * 2022-06-30 2024-01-04 复旦大学 Her2纳米抗体及偶联物的制备方法和用途

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009068625A3 (en) * 2007-11-27 2009-08-06 Ablynx Nv Amino acid sequences directed against her2 and polypeptides comprising the same for the treatment of cancers and/or tumors
CN102321175A (zh) * 2011-09-21 2012-01-18 天津胜发生物技术有限公司 针对乳腺癌Her2/new的纳米抗体或多肽

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009068625A3 (en) * 2007-11-27 2009-08-06 Ablynx Nv Amino acid sequences directed against her2 and polypeptides comprising the same for the treatment of cancers and/or tumors
CN102321175A (zh) * 2011-09-21 2012-01-18 天津胜发生物技术有限公司 针对乳腺癌Her2/new的纳米抗体或多肽

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JAMNAN F. R.等: "《Targeting high affinity and epitope-distinct oligoclonal nanobodies to HER2 over-expressing tumor cells》", 《EXPERIMENTAL CELL RESEARCH》 *
邢美宁: "《重组人HER2胞外域的制备及其特异性VHH纳米抗体的筛选》", 《中国优秀硕士学位论文》 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018233624A1 (zh) * 2017-06-20 2018-12-27 和迈生物科技有限公司 抗Her2纳米抗体及其编码序列和用途
US11517632B2 (en) 2017-06-20 2022-12-06 Nanomab Technology Limited Anti-Her2 single chain antibody and coding sequence and use thereof
CN109627319B (zh) * 2019-01-11 2022-06-21 北京协同创新研究院 一种抗her-2的重链抗体及其应用
CN109627319A (zh) * 2019-01-11 2019-04-16 北京协同创新研究院 一种抗her-2的重链抗体及其应用
CN110950967A (zh) * 2019-12-13 2020-04-03 山东民康生物科技有限公司 抗人血清白蛋白纳米抗体与il-2融合蛋白及制备方法
CN110950967B (zh) * 2019-12-13 2022-05-13 山东民康生物科技有限公司 抗人血清白蛋白纳米抗体与il-2融合蛋白及制备方法
CN114085287A (zh) * 2021-11-12 2022-02-25 中国农业科学院北京畜牧兽医研究所 犬细小病毒纳米抗体cpv-vhh-f5及其应用
CN114085287B (zh) * 2021-11-12 2022-10-25 中国农业科学院北京畜牧兽医研究所 犬细小病毒纳米抗体cpv-vhh-f5及其应用
CN114409787A (zh) * 2022-02-18 2022-04-29 南京英瀚斯生物科技有限公司 一种针对her3纳米抗体的分离纯化方法
WO2024001844A1 (zh) * 2022-06-30 2024-01-04 复旦大学 Her2纳米抗体及偶联物的制备方法和用途
WO2024001510A1 (zh) * 2022-06-30 2024-01-04 复旦大学 Her2纳米抗体及偶联物的制备方法和用途
CN116574186A (zh) * 2023-07-07 2023-08-11 云南大学 特异性结合her2的纳米抗体及其应用
CN116574186B (zh) * 2023-07-07 2023-11-28 云南大学 特异性结合her2的纳米抗体及其应用

Similar Documents

Publication Publication Date Title
CN106866823A (zh) 一种抗Her2的纳米抗体
CN106939047A (zh) 一种pd-l1抗体及其制备方法
CN108659129A (zh) 一种抗gpc3蛋白的纳米抗体及其制备方法和应用
CN102321175B (zh) 针对乳腺癌Her2/new的纳米抗体或多肽
KR20190113876A (ko) 개선된 항vegfr-2 모노클로널 항체
CN111825767B (zh) 一种人表皮生长因子受体2的单域抗体、检测试剂盒及其应用
CN108530538B (zh) EpCAM单域抗体E6
CN101062949B (zh) 重组抗人IgE单克隆抗体及其制备方法和用途
CN111234023B (zh) 小细胞肺癌检测试剂盒
CN111551732A (zh) 定量检测体液中肿瘤来源细胞外囊泡pd-l1含量的方法、elisa试剂盒及使用方法
WO2024124637A1 (zh) 抗cll1单域抗体及其应用
CN108586614B (zh) EpCAM单域抗体D7
CN113501874B (zh) 一种抗人cd47蛋白的抗体、检测试剂盒及其应用
JPH07506727A (ja) 凝集アッセイ用試薬
CN103342752A (zh) 一种抗人组织因子单链抗体及其制备方法
CN108003239B (zh) 一种全人源抗egfr单链抗体及其应用
CN109652555A (zh) 一种诊治恶性肿瘤的分子标志物
CN107163131B (zh) 肿瘤抑制因子p16的抗原多肽和其应用
CN108129564B (zh) 一种全人源的抗vegf单链抗体及其应用
CN105237637B (zh) 抗人神经纤毛蛋白1的单域抗体及其制备方法
CN112724253B (zh) 抗人穹窿体蛋白的抗体及其应用
CN116813774B (zh) 一种抗人cd47蛋白的抗体、核酸分子及其应用
CN103044547B (zh) 一种抗人源多药耐药蛋白Fab抗体及其制备方法
CN115611982B (zh) 一种抗人MICA/Bα3区的单克隆抗体及其应用
CN114891126B (zh) 一种靶向fshr和cd3分子的双结合蛋白及其应用

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
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20170620

WD01 Invention patent application deemed withdrawn after publication