CN114349860A - 一种抗成纤维细胞生长因子受体4单克隆抗体1b5及其制备方法和用途 - Google Patents
一种抗成纤维细胞生长因子受体4单克隆抗体1b5及其制备方法和用途 Download PDFInfo
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Abstract
本发明涉及一种抗成纤维细胞生长因子受体4抗体1B5及其制备方法和用途，具体公开了抗成纤维细胞生长因子受体4抗体或其抗原结合片段，其具有如SEQ ID NO:18、SEQ ID NO:19和SEQ ID NO:20所示的三个重链互补决定区(CDR)，并具有如SEQ ID NO:26、SEQ ID NO:27和SEQ ID NO:28所示的三个轻链互补决定区。还公开了一种核苷酸序列，其特征在于：其编码如前述的抗人成纤维细胞生长因子受体4的单克隆抗体或其抗原结合片段；以及单克隆抗体作为检测试剂的用途。该抗体具有灵敏度高，特异性好等优点。
Description
技术领域
本发明涉及抗体领域，具体涉及一种抗成纤维细胞生长因子受体4(fibroblastgrowth factor receptor，FGFR4)单克隆抗体及其制备方法和用途。
背景技术
成纤维细胞生长因子受体4(fibroblast growth factor receptor，FGFR4)是一种在特定组织特异性表达的酪氨酸激酶受体，它参与调节细胞的生长、繁殖和移动过程，也能够调节胆汁酸和脂质代谢。FGFR4及其相关分子的基因变异和异常表达导致癌症的发生发展、增殖、存活及转移。FGFR4高表达于人类多种癌症，包括乳腺癌、膀胱癌、肺癌、前列腺癌、胃癌、肝癌、胰腺癌、脑胶质瘤等等多种恶性肿瘤。FGFR4单克隆抗体能特异性地阻止配体受体结合及受体二聚化，从而直接抑制癌症进展。同时抗体可以结合毒素和放射性同位素等分子，为肿瘤细胞靶向化疗或放疗提供了可能。由于抗体抗原识别的高度特异性，以FGFR4为靶点的单克隆抗体将大大降低治疗副反应。
作为生物学研究领域应用最广泛的工具，抗体产品的地位毋庸置疑。而抗体的特异性和应用范围长久以来困扰着抗体产业，也是整个生物学研究领域的极大危机。抗体产品糟糕的质量会直接导致实验结果的误差，以及研究结果无法被重复和再现。研究项目的进展往往会因为抗体带来的问题停滞不前，由此造成的损失也是惊人的。据2011年的数据统计，仅在美国，平均每年就有3.5亿美元被浪费在这些无效抗体上。而在世界范围内，每年竟有8亿美元被浪费，占到了全球科研抗体总开销的50％。
发明内容
为了克服上述问题，本发明提供一种抗FGFR4的单克隆抗体1B5，其是特异性靶向肝癌细胞的FGFR4分子，可以应用于免疫印迹，流式细胞技术和ELISA等实验，是研究FGFR4功能的有力工具。
本发明一个方面提供了一种分离的抗成纤维细胞生长因子受体4的抗体或其抗原结合片段，其具有如SEQ ID NO:2、SEQ ID NO:3和SEQ ID NO:4所示的三个重链互补决定区(CDR)，并具有如SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12所示的三个轻链互补决定区。
本发明另个方面提供了一种分离的抗成纤维细胞生长因子受体4的抗体或其抗原结合片段，其具有如SEQ ID No：1所示的重链可变区，如SEQ ID No:9所示的轻链可变区。
本发明再一个方面提供了一种核苷酸序列，其特征在于：其编码如前述的抗人成纤维细胞生长因子受体4的单克隆抗体或其抗原结合片段。
在本发明的技术方案中，所述的抗体为单克隆抗体。
本发明再一个方面提供了一种重组载体，其特征在于：包含前述的核苷酸序列。
本发明再一个方面提供了一种宿主细胞，其特征在于：包含前述载体或载体组，优选地，所述宿主细胞是原核的或真核的，更优选的选自酵母细胞、哺乳动物细胞或适用于制备抗体或其抗原结合片段的其它细胞。
本发明再一个方面提供了一种试剂盒，所述试剂盒包含如前述的抗体或其抗原结合片段。
本发明再一个方面提供了一种检测试剂，所述检测试剂包含如前述的抗体或其抗原结合片段。
本发明再一个方面提供了上述抗体或其抗原结合片段作为检测试剂的用途，所述试剂用于以下用途的试剂：酶联免疫吸附检测(ELISA)、免疫印迹(Western Blot)、流式细胞术(FACS)、免疫组织化学(IHC)检测或者免疫PCR。
在上述在免疫学检测中，抗体或其抗原结合片段可单独或与通过化学键偶联，静电吸附或者亲疏水性吸附，而连接缀合物包括辣根过氧化物酶(HRP)，碱性磷酸酶(AP)，生物素(Biotin)，异硫氰酸荧光素(FITC)，Cy3、Cy5、磁珠和琼脂糖等缀合物连接。
在本发明的技术方案中，检测试剂可用于非诊断的治疗目的检测。
本发明再一个方面提供了上述抗体或其抗原结合片段作为体外分离或纯化人成纤维细胞生长因子受体4的试剂的用途。
在本发明中，上述抗体或其抗原结合片段通过杂交瘤方法制备。
在本发明中，所述抗体或其抗原结合片段的重链和轻链可变区的核苷酸和氨基酸序列如下所示。
重链可变区氨基酸序列：
EVMLVQSGPELKKPGETVKISCKASGYTFTNFGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFVFSLETSASTAYLQINNLKNEDTATYLCVRSSSGYVFDYWGQGTIVTVG
SEQ ID NO.1
其中，重链可变区CDR1氨基酸序列为：GYTFTNFG SEQ ID NO.2
重链可变区CDR2氨基酸序列为：INTYTGEP SEQ ID NO.3
重链可变区CDR3氨基酸序列为：VRSSSGYVFDY SEQ ID NO.4
重链可变区FR1氨基酸序列为：EVMLVQSGPELKKPGETVKISCKAS SEQ ID NO.5
重链可变区FR2氨基酸序列为：MNWVKQAPGKGLKWMGW SEQ ID NO.6
重链可变区FR3氨基酸序列为：TYADDFKGRFVFSLETSASTAYLQINNLKNEDTATYLC SEQID NO.7
重链可变区FR4氨基酸序列为：WGQGTIVTVG SEQ ID NO.8
轻链可变区氨基酸序列：DIVMTQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLLTFGEGTKLEIT SEQ ID NO.9
其中，轻链可变区CDR1氨基酸序列为：QSLLNSRTRKNY SEQ ID NO.10
轻链可变区CDR2氨基酸序列为：WAS SEQ ID NO.11
轻链可变区CDR3氨基酸序列为：KQSYNLLT SEQ ID NO.12
轻链可变区FR1氨基酸序列为：DIVMTQSPSSLAVSAGEKVTMSCKSS SEQ ID NO.13
轻链可变区FR2氨基酸序列为：LAWYQQKPGQSPKLLIY SEQ ID NO.14
轻链可变区FR3氨基酸序列为：TRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC
SEQ ID NO.15
轻链可变区FR4氨基酸序列为：FGEGTKLEIT SEQ ID NO.16
重链可变区核苷酸序列：GAGGTGATGCTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGTAAGGCTTCTGGGTATACCTTCACAAACTTTGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGACGGTTTGTCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAATCTCAAAAATGAGGACACGGCTACATATCTCTGTGTAAGATCCAGCTCGGGCTACGTTTTTGACTACTGGGGCCAAGGAACTATAGTCACAGTCGGT
SEQ ID NO.17
重链可变区CDR1核苷酸序列：GGGTATACCTTCACAAACTTTGGA SEQ ID NO.18
重链可变区CDR2核苷酸序列：ATAAACACCTACACTGGAGAGCCA SEQ ID NO.19
重链可变区CDR3核苷酸序列：GTAAGATCCAGCTCGGGCTACGTTTTTGACTACSEQ IDNO.20
重链可变区FR1核苷酸序列：GAGGTGATGCTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGTAAGGCTTCT SEQ ID NO.21
重链可变区FR2核苷酸序列：：ATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGG SEQ ID NO.22
重链可变区FR3核苷酸序列：ACATATGCTGATGACTTCAAGGGACGGTTTGTCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAATCTCAAAAATGAGGACACGGCTACATATCTCTGT
SEQ ID NO.23
重链可变区FR4核苷酸序列：TGGGGCCAAGGAACTATAGTCACAGTCGGT SEQ ID NO.24
轻链可变区核苷酸序列：GACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGAACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGCAAGCAATCTTATAATCTGCTCACGTTCGGAGAAGGCACAAAACTGGAAATAACC SEQ ID NO.25
轻链可变区CDR1核苷酸序列：CAGAGTCTGCTCAACAGTAGAACCCGAAAGAACTAC SEQ IDNO.26
轻链可变区CDR2核苷酸序列：TGGGCATCC SEQ ID NO.27
轻链可变区CDR3核苷酸序列：AAGCAATCTTATAATCTGCTCACG SEQ ID NO.28
轻链可变区FR1核苷酸序列：GACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAATCCAGT SEQ ID NO.29
轻链可变区FR2核苷酸序列：TTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTAC SEQ ID NO.30
轻链可变区FR3核苷酸序列：ACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGC SEQ ID NO.31
轻链可变区FR4核苷酸序列：TTCGGAGAAGGCACAAAACTGGAAATAACC SEQ ID NO.32
重链可变区基因全长351bp，编码氨基酸残基117个。重链可变区核苷酸序列如SEQID NO:17所示，重链可变区氨基酸序列如SEQ ID NO:1所示，重链CDR1氨基酸序列如SEQ IDNo:2所示，重链CDR 2氨基酸序列如SEQ ID No:3所示，重链CDR3氨基酸序列如SEQ ID No:4所示。
轻链可变区基因序列全长336bp，编码氨基酸残基112个。轻链可变区核苷酸序列如SEQ ID NO:25所示，轻链可变区氨基酸序列如SEQ ID NO:9，轻链CDR 1氨基酸序列如SEQID No:10所示，轻链CDR 2氨基酸序列如SEQ ID No:11所示，轻链CDR 3氨基酸序列如SEQID No:12所示。
有益效果
本发明提供了一种抗人成纤维细胞生长因子受体4的单克隆抗体的制备方法、鉴定及应用，该单克隆抗体具有灵敏度高，特异性好等优点，适用于不同检测方法。本发明提供的单克隆抗体，可以广泛用于Western blot、ELISA、flowCytometry等不同手段检测人成纤维细胞生长因子受体4，为研究人成纤维细胞生长因子受体4的功能提供了基础。
附图说明
图1为1B5抗体与FGFR4的亲和力结果图。
图2为实施例4中1B5在流式细胞检测中应用的结果。
图3为实施例4中1B5在ELISA检测中应用的结果。
具体实施方式
为了使本发明的上述目的、特征和优点能够更加明显易懂，下面对本发明的具体实施方式做详细的说明，但不能理解为对本发明的可实施范围的限定。
实施例1FGFR4单克隆抗体的生产
采用杂交瘤法(由Kohler等，Nature,256:495(1975)首先提出)。用FGFR4蛋白抗原(购自义翘神州)免疫雌性BALB/c小鼠(6周龄)，首次免疫使用弗氏完全佐剂进行乳化抗原，第二次免疫开始，使用弗氏不完全佐剂乳化抗原，皮下5～6点注射，每只小鼠注射的抗原量为～100ug。在第3次免疫后10天，对小鼠剪尾采集少量血液进行血清效价ELISA检测，选择抗体滴度高(>1:100000)的小鼠进行加强第4次免疫，通过腹腔注射抗原蛋白，每只小鼠注射100ug。第4次免疫后3～5天，处死小鼠取其脾细胞与SP2/0细胞融合，通过HAT培养基培养获得稳定的杂交瘤细胞。通过ELISA法筛选得到能分泌FGFR4抗体的杂交瘤细胞，通过有限稀释的方法进行亚克隆，ELISA法筛选得到能分泌FGFR4抗体的单克隆杂交瘤细胞株，通过逐级扩大培养，液氮冻存保种。
腹水抗体制备：雌性BALB/c小鼠(8周龄)腹腔注射弗氏不完全佐剂，每只小鼠注射0.5mL，3～5天后腹腔注射处于对数生长期的杂交瘤细胞，每只小鼠注射1～5×105个细胞(0.5mL)，注射杂交瘤细胞～11天后处死小鼠，获得腹水。3000rpm，4℃离心10min，去除沉淀，用10倍体积1×PB溶液稀释腹水，混匀后过0.45μm滤膜。通过Protein G(Protein GSepharose 4Fast Flow,GE Healthcare)亲和纯化腹水,得到纯化的FGFR4抗体蛋白，用BCA法测抗体浓度。将纯化抗体跑SDS-PAGE(上样量5.4μg)，考马斯亮蓝染色。
实施例2FGFR4单克隆抗体杂交瘤细胞的抗体基因测序
收获处于对数生长期的单克隆抗体杂交瘤细胞，TRIZOL裂解进行RNA提取，反转录后获得cDNA，扩增并获得重链和轻链可变区，非功能性VK基因去除，克隆至pMD18-T载体，测序，使用IMGT/V-QUEST数据库进行测序结果比对，进一步分析。
单克隆抗体1B5重链蛋白序列：
EVMLVQSGPELKKPGETVKISCKASGYTFTNFGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFVFSLETSASTAYLQINNLKNEDTATYLCVRSSSGYVFDYWGQGTIVTVG SEQ ID NO.1
其中，重链CDR1氨基酸序列为：GYTFTNFG SEQ ID NO.2
重链CDR2氨基酸序列为：INTYTGEP SEQ ID NO.3
重链CDR3氨基酸序列为：VRSSSGYVFDY SEQ ID NO.4
重链FR1氨基酸序列为：EVMLVQSGPELKKPGETVKISCKAS SEQ ID NO.5
重链FR2氨基酸序列为：MNWVKQAPGKGLKWMGW SEQ ID NO.6
重链FR3氨基酸序列为：TYADDFKGRFVFSLETSASTAYLQINNLKNEDTATYLC SEQ IDNO.7
重链FR4氨基酸序列为：WGQGTIVTVG SEQ ID NO.8
单克隆抗体1B5轻链蛋白序列：
DIVMTQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLLTFGEGTKLEIT SEQ ID NO.9
其中，轻链CDR1氨基酸序列为：QSLLNSRTRKNY SEQ ID NO.10
轻链CDR2氨基酸序列为：WAS SEQ ID NO.11
轻链CDR3氨基酸序列为：KQSYNLLT SEQ ID NO.12
轻链FR1氨基酸序列为：DIVMTQSPSSLAVSAGEKVTMSCKSS SEQ ID NO.13
轻链FR2氨基酸序列为：LAWYQQKPGQSPKLLIY SEQ ID NO.14
轻链FR3氨基酸序列为：TRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC SEQ ID NO.15
轻链FR4氨基酸序列为：FGEGTKLEIT SEQ ID NO.16
单克隆抗体1B5重链核苷酸序列：
GAGGTGATGCTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGTAAGGCTTCTGGGTATACCTTCACAAACTTTGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGACGGTTTGTCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAATCTCAAAAATGAGGACACGGCTACATATCTCTGTGTAAGATCCAGCTCGGGCTACGTTTTTGACTACTGGGGCCAAGGAACTATAGTCACAGTCGGT
SEQ ID NO.17
其中，重链CDR1核苷酸序列为：GGGTATACCTTCACAAACTTTGGA SEQ ID NO.18
重链CDR2核苷酸序列为：ATAAACACCTACACTGGAGAGCCA SEQ ID NO.19
重链CDR3核苷酸序列为：GTAAGATCCAGCTCGGGCTACGTTTTTGACTAC SEQ ID NO.20
重链FR1核苷酸序列为：GAGGTGATGCTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGTAAGGCTTCT SEQ ID NO.21
重链FR2核苷酸序列为：ATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGG SEQ ID NO.22
重链FR3核苷酸序列为：ACATATGCTGATGACTTCAAGGGACGGTTTGTCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAATCTCAAAAATGAGGACACGGCTACATATCTCTGT
SEQ ID NO.23
重链FR4核苷酸序列为：TGGGGCCAAGGAACTATAGTCACAGTCGGT SEQ ID NO.24
单克隆抗体1B5轻链核苷酸序列：
GACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGAACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGCAAGCAATCTTATAATCTGCTCACGTTCGGAGAAGGCACAAAACTGGAAATAACC SEQ ID NO.25
其中，轻链CDR1核苷酸序列为：CAGAGTCTGCTCAACAGTAGAACCCGAAAGAACTAC SEQ IDNO.26
轻链CDR2核苷酸序列为：TGGGCATCC SEQ ID NO.27
轻链CDR3核苷酸序列为：AAGCAATCTTATAATCTGCTCACG SEQ ID NO.28
轻链FR1核苷酸序列为：GACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAATCCAGT SEQ ID NO.29
轻链FR2核苷酸序列为：TTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTAC SEQ ID NO.30
轻链FR3核苷酸序列为：ACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGC SEQ ID NO.31
轻链FR4核苷酸序列为：TTCGGAGAAGGCACAAAACTGGAAATAACC SEQ ID NO.32。
实施例3FGFR4单克隆抗体的亲和力测定
使用10ug/mL FGFR4蛋白与传感器进行固化结合，使用SD buffer(PBS+0.02％Tween 20+0.1％BSA)配制不同浓度的1B5抗体(566.7nM、283.3nM、141.7nM、70.8nM、35.4nM、17.7nM、8.84nM)作为流动相，使用分子间相互作用仪(OCTET K2，PALL lifescience)进行亲和力检测，程序设定为：Baseline 240s，Loading 360s，Baseline2 180s，Association 480s，Dissociation 480s，使用AHC(Anti-hIgG Fc Capture)传感器。结果如图1所示，1B5抗体与FGFR4的亲和力<1.0E-12(M)。
实施例4FGFR4单克隆抗体的应用
4.1FGFR4单克隆抗体的流式检测应用
1)收集HepG2细胞，吹散计数，分装5个离心管，每管3×105个细胞，500g离心5min，去上清，分别加入1mL PBS，500g离心5min，去上清。
2)按下表加入试剂：
放入冰上，孵育，30min。
3)各加入1mL PBS+5％FBS，500g离心5min，去上清，各加入100μL DMEM培养液+1μLPE goat anti-mouse lgG(minimal x-reactivity)antibody(biolegend,405307)，冰上孵育20min。
4)各加入1mL PBS+5％FBS，500g离心5min，去上清。
5)每管加入300μL PBS+5％FBS，重悬，使用贝克曼流式细胞仪(Beckman CytoFLEXCM)检测。
如图2所示，1B5抗体能与HepG2细胞表面的FGFR4蛋白结合，应用于流式细胞检测，且检测灵敏度优于商品化阳性对照。
4.2FGFR4单克隆抗体的ELISA检测应用
酶标板每孔包被100ng FGFR4蛋白，4℃包被过夜，经封闭洗板后，加入的1B5抗体，浓度为1μg/mL，每孔100μL，37度孵育1h。PBST洗板5次，加入1:5000稀释的HRP-羊抗小鼠IgG抗体，37℃孵育45min。PBST洗板5次，每孔加入100μL TMB，室温避光反应3min，每孔加入50μL终止液终止，酶标仪测OD450。如图3所示，1B5能用于ELISA检测应用。
4.3FGFR4单克隆抗体的Western blot检测应用
FGFR4蛋白加5×loading buffer混合后，煮沸变性6min，跑SDS-PAGE，每孔上样20μL，～500ng蛋白/孔，400mA转膜1h，5％脱脂奶粉室温封闭1h，加入1B5抗体(3mL 5％脱脂奶粉中加入～10ug抗体)4度孵育过夜，PBST洗3遍后加入1:5000稀释羊抗鼠IgG二抗，室温孵育45min，PBST洗3遍后加入Western HRP底物(密理博，WBLUR0500)显影。
如图所示，1B5能用于Western blot检测应用。
SEQUENCE LISTING
<110> 深圳宾德生物技术有限公司
<120> 一种抗成纤维细胞生长因子受体4单克隆抗体1B5及其制备方法和用途
<130> CP121010737C
<160> 32
<170> PatentIn version 3.3
<210> 1
<211> 117
<212> PRT
<213> 人工序列
<400> 1
Glu Val Met Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Phe
20 25 30
Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Leu Cys
85 90 95
Val Arg Ser Ser Ser Gly Tyr Val Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Ile Val Thr Val Gly
115
<210> 2
<211> 8
<212> PRT
<213> 人工序列
<400> 2
Gly Tyr Thr Phe Thr Asn Phe Gly
1 5
<210> 3
<211> 8
<212> PRT
<213> 人工序列
<400> 3
Ile Asn Thr Tyr Thr Gly Glu Pro
1 5
<210> 4
<211> 11
<212> PRT
<213> 人工序列
<400> 4
Val Arg Ser Ser Ser Gly Tyr Val Phe Asp Tyr
1 5 10
<210> 5
<211> 25
<212> PRT
<213> 人工序列
<400> 5
Glu Val Met Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser
20 25
<210> 6
<211> 17
<212> PRT
<213> 人工序列
<400> 6
Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly
1 5 10 15
Trp
<210> 7
<211> 38
<212> PRT
<213> 人工序列
<400> 7
Thr Tyr Ala Asp Asp Phe Lys Gly Arg Phe Val Phe Ser Leu Glu Thr
1 5 10 15
Ser Ala Ser Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp
20 25 30
Thr Ala Thr Tyr Leu Cys
35
<210> 8
<211> 10
<212> PRT
<213> 人工序列
<400> 8
Trp Gly Gln Gly Thr Ile Val Thr Val Gly
1 5 10
<210> 9
<211> 112
<212> PRT
<213> 人工序列
<400> 9
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln
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Ser Tyr Asn Leu Leu Thr Phe Gly Glu Gly Thr Lys Leu Glu Ile Thr
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<210> 10
<211> 12
<212> PRT
<213> 人工序列
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Gln Ser Leu Leu Asn Ser Arg Thr Arg Lys Asn Tyr
1 5 10
<210> 11
<211> 3
<212> DNA
<213> 人工序列
<400> 11
was 3
<210> 12
<211> 8
<212> PRT
<213> 人工序列
<400> 12
Lys Gln Ser Tyr Asn Leu Leu Thr
1 5
<210> 13
<211> 26
<212> PRT
<213> 人工序列
<400> 13
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser
20 25
<210> 14
<211> 17
<212> PRT
<213> 人工序列
<400> 14
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
1 5 10 15
Tyr
<210> 15
<211> 36
<212> PRT
<213> 人工序列
<400> 15
Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly
1 5 10 15
Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala
20 25 30
Val Tyr Tyr Cys
35
<210> 16
<211> 10
<212> PRT
<213> 人工序列
<400> 16
Phe Gly Glu Gly Thr Lys Leu Glu Ile Thr
1 5 10
<210> 17
<211> 351
<212> DNA
<213> 人工序列
<400> 17
gaggtgatgc tggtgcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc 60
tcctgtaagg cttctgggta taccttcaca aactttggaa tgaactgggt gaagcaggct 120
ccaggaaagg gtttaaagtg gatgggctgg ataaacacct acactggaga gccaacatat 180
gctgatgact tcaagggacg gtttgtcttc tctttggaaa cctctgccag cactgcctat 240
ttgcagatca acaatctcaa aaatgaggac acggctacat atctctgtgt aagatccagc 300
tcgggctacg tttttgacta ctggggccaa ggaactatag tcacagtcgg t 351
<210> 18
<211> 24
<212> DNA
<213> 人工序列
<400> 18
gggtatacct tcacaaactt tgga 24
<210> 19
<211> 24
<212> DNA
<213> 人工序列
<400> 19
ataaacacct acactggaga gcca 24
<210> 20
<211> 33
<212> DNA
<213> 人工序列
<400> 20
gtaagatcca gctcgggcta cgtttttgac tac 33
<210> 21
<211> 75
<212> DNA
<213> 人工序列
<400> 21
gaggtgatgc tggtgcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc 60
tcctgtaagg cttct 75
<210> 22
<211> 51
<212> DNA
<213> 人工序列
<400> 22
atgaactggg tgaagcaggc tccaggaaag ggtttaaagt ggatgggctg g 51
<210> 23
<211> 114
<212> DNA
<213> 人工序列
<400> 23
acatatgctg atgacttcaa gggacggttt gtcttctctt tggaaacctc tgccagcact 60
gcctatttgc agatcaacaa tctcaaaaat gaggacacgg ctacatatct ctgt 114
<210> 24
<211> 30
<212> DNA
<213> 人工序列
<400> 24
tggggccaag gaactatagt cacagtcggt 30
<210> 25
<211> 336
<212> DNA
<213> 人工序列
<400> 25
gacattgtga tgacccagtc tccatcctcc ctggctgtgt cagcaggaga gaaggtcact 60
atgagctgca aatccagtca gagtctgctc aacagtagaa cccgaaagaa ctacttggct 120
tggtaccagc agaaaccagg gcagtctcct aaactgctga tctactgggc atccactagg 180
gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240
atcagcagtg tgcaggctga agacctggca gtttattact gcaagcaatc ttataatctg 300
ctcacgttcg gagaaggcac aaaactggaa ataacc 336
<210> 26
<211> 36
<212> DNA
<213> 人工序列
<400> 26
cagagtctgc tcaacagtag aacccgaaag aactac 36
<210> 27
<211> 9
<212> DNA
<213> 人工序列
<400> 27
tgggcatcc 9
<210> 28
<211> 24
<212> PRT
<213> 人工序列
<400> 28
Ala Ala Gly Cys Ala Ala Thr Cys Thr Thr Ala Thr Ala Ala Thr Cys
1 5 10 15
Thr Gly Cys Thr Cys Ala Cys Gly
20
<210> 29
<211> 78
<212> DNA
<213> 人工序列
<400> 29
gacattgtga tgacccagtc tccatcctcc ctggctgtgt cagcaggaga gaaggtcact 60
atgagctgca aatccagt 78
<210> 30
<211> 51
<212> DNA
<213> 人工序列
<400> 30
ttggcttggt accagcagaa accagggcag tctcctaaac tgctgatcta c 51
<210> 31
<211> 108
<212> DNA
<213> 人工序列
<400> 31
actagggaat ctggggtccc tgatcgcttc acaggcagtg gatctgggac agatttcact 60
ctcaccatca gcagtgtgca ggctgaagac ctggcagttt attactgc 108
<210> 32
<211> 30
<212> DNA
<213> 人工序列
<400> 32
ttcggagaag gcacaaaact ggaaataacc 30
Claims (10)
1.一种分离的抗成纤维细胞生长因子受体4的抗体或其抗原结合片段，其特征在于：其具有如SEQ ID NO:2、SEQ ID NO:3和SEQ ID NO:4所示的三个重链互补决定区，并具有如SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12所示的三个轻链互补决定区；
其中，重链互补决定区CDR1如SEQ ID NO:2所示；
重链互补决定区CDR2如SEQ ID NO:3所示；
重链互补决定区CDR3如SEQ ID NO:4所示；
轻链互补决定区CDR1如SEQ ID NO:10所示；
轻链互补决定区CDR2如SEQ ID NO:11所示；
轻链互补决定区CDR3如SEQ ID NO:12所示。
2.一种分离的抗成纤维细胞生长因子受体4的抗体或其抗原结合片段，其特征在于：其具有如SEQ ID No:1所示的重链可变区，如SEQ ID No:9所示的轻链可变区。
3.一种核苷酸，其特征在于：其编码如权利要求1或2所述的抗成纤维细胞生长因子受体4的抗体或其抗原结合片段。
4.一种重组载体，其特征在于：包含权利要求3所述的核苷酸序列。
5.一种宿主细胞，其特征在于：包含权利要求4所述的重组载体；
优选地，所述宿主细胞是原核的或真核的；
优选地，所述宿主细胞选自酵母细胞、哺乳动物细胞或适用于制备抗体或其抗原结合片段的其它细胞。
6.一种试剂盒，其特征在于：所述试剂盒包含如权利要求1或2所述的抗体或其抗原结合片段。
7.一种检测试剂，其特征在于：所述检测试剂包含如权利要求1或2所述的抗体或其抗原结合片段；
优选地，所述检测试剂用于酶联免疫吸附检测、免疫印迹、流式细胞术、免疫组织化学检测或者免疫PCR。
8.权利要求1或2所述的抗体或其抗原结合片段在制备检测人成纤维细胞生长因子受体4的试剂中的用途。
9.根据权利要求8所述的用途，所述试剂用于以下用途的试剂：酶联免疫吸附检测、免疫印迹、流式细胞术、免疫组织化学检测或者免疫PCR。
10.权利要求1或2所述的抗体或其抗原结合片段在制备体外分离或纯化人成纤维细胞生长因子受体4的试剂中的用途。
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| WO2021203700A1 (zh) * | 2020-04-08 | 2021-10-14 | 中国科学院深圳先进技术研究院 | 一种抗成纤维细胞生长因子受体4单克隆抗体及其制备方法和用途 |
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| WO2021203700A1 (zh) * | 2020-04-08 | 2021-10-14 | 中国科学院深圳先进技术研究院 | 一种抗成纤维细胞生长因子受体4单克隆抗体及其制备方法和用途 |
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