WO2021203700A1 - 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 - Google Patents

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 Download PDF

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WO2021203700A1
WO2021203700A1 PCT/CN2020/129152 CN2020129152W WO2021203700A1 WO 2021203700 A1 WO2021203700 A1 WO 2021203700A1 CN 2020129152 W CN2020129152 W CN 2020129152W WO 2021203700 A1 WO2021203700 A1 WO 2021203700A1
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seq
antibody
variable region
antigen
chain variable
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PCT/CN2020/129152
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French (fr)
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万晓春
李俊鑫
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中国科学院深圳先进技术研究院
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    • 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
    • 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
    • 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
    • C07K2317/565—Complementarity determining region [CDR]

Definitions

  • the present invention relates to the field of antibodies, in particular to an anti-fibroblast growth factor receptor 4 (fibroblast growth factor receptor 4) receptor, FGFR4) monoclonal antibody and its preparation method and use.
  • fibroblast growth factor receptor 4 fibroblast growth factor receptor 4
  • FGFR4 FGFR4 monoclonal antibody
  • Fibroblast growth factor receptor 4 (fibroblast growth factor receptor 4) Receptor, FGFR4) is a tyrosine kinase receptor that is specifically expressed in specific tissues. It is involved in the regulation of cell growth, reproduction and movement, and can also regulate bile acid and lipid metabolism. The gene mutation and abnormal expression of FGFR4 and its related molecules lead to the development, proliferation, survival and metastasis of cancer. FGFR4 is highly expressed in a variety of human cancers, including breast cancer, bladder cancer, lung cancer, prostate cancer, gastric cancer, liver cancer, pancreatic cancer, glioma and many other malignant tumors. FGFR4 monoclonal antibody can specifically prevent ligand-receptor binding and receptor dimerization, thereby directly inhibiting cancer progression.
  • antibodies can bind to toxins and radioisotopes and other molecules, providing the possibility of targeted chemotherapy or radiotherapy for tumor cells. Due to the high specificity of antibody antigen recognition, monoclonal antibodies targeting FGFR4 will greatly reduce the side effects of treatment.
  • the present invention provides an anti-FGFR4 monoclonal antibody 6G12F5, which is a FGFR4 molecule that specifically targets liver cancer cells. It can be applied to experiments such as immunoblotting, flow cytometry and ELISA. It is used to study the function of FGFR4. Powerful tool.
  • One aspect of the present invention provides an isolated anti-fibroblast growth factor receptor 4 antibody or antigen-binding fragment thereof, which has as shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 Three heavy chain complementarity determining regions (CDRs), and three light chain complementarity determining regions as shown in SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.
  • SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 Three heavy chain complementarity determining regions (CDRs), and three light chain complementarity determining regions as shown in SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.
  • Another aspect of the present invention provides an isolated anti-fibroblast growth factor receptor 4 antibody or antigen-binding fragment thereof, which has a heavy chain variable region as shown in SEQ ID No: 17, as shown in SEQ ID No: The variable region of the light chain shown at 25.
  • Another aspect of the present invention provides a nucleotide sequence characterized in that it encodes the aforementioned anti-human fibroblast growth factor receptor 4 monoclonal antibody or antigen-binding fragment thereof.
  • the antibody is a monoclonal antibody.
  • Another aspect of the present invention provides a recombinant vector, which is characterized by comprising the aforementioned nucleotide sequence.
  • Another aspect of the present invention provides a host cell characterized by comprising the aforementioned vector or vector group.
  • the host cell is prokaryotic or eukaryotic, more preferably selected from yeast cells, mammalian cells or It is applicable to other cells for preparing antibodies or antigen-binding fragments thereof.
  • kits comprising the aforementioned antibody or antigen-binding fragment thereof.
  • Another aspect of the present invention provides a detection reagent, which comprises the aforementioned antibody or antigen-binding fragment thereof.
  • Another aspect of the present invention provides the use of the above-mentioned antibody or antigen-binding fragment thereof as a detection reagent.
  • the reagent is used for the following purposes: enzyme-linked immunosorbent assay (ELISA), western blotting Blot), flow cytometry (FACS), immunohistochemistry (IHC) detection or immuno-PCR.
  • ELISA enzyme-linked immunosorbent assay
  • FACS flow cytometry
  • IHC immunohistochemistry
  • the antibody or its antigen-binding fragment can be singly or coupled with chemical bonds, electrostatic adsorption or hydrophilic adsorption, and the linking conjugate includes horseradish peroxidase (HRP), alkaline phosphatase (AP), Biotin (Biotin), Fluorescein isothiocyanate (FITC), Cy3, Cy5, magnetic beads and agarose and other conjugates are connected.
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • Biotin Biotin
  • FITC Fluorescein isothiocyanate
  • Cy3, Cy5 magnetic beads and agarose and other conjugates are connected.
  • the detection reagent can be used for non-diagnostic treatment purposes.
  • Another aspect of the present invention provides the use of the above-mentioned antibody or antigen-binding fragment thereof as a reagent for isolating or purifying human fibroblast growth factor receptor 4 in vitro.
  • the above-mentioned antibody or antigen-binding fragment thereof is prepared by the hybridoma method.
  • nucleotide and amino acid sequences of the heavy chain and light chain variable regions of the antibody or its antigen-binding fragment are shown below.
  • the light chain variable region nucleotide sequence GACATCCAGATGACTCAGACTCCAGCCTCCCTATCTGCATCTGTGGAAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACAGTTATTTAGCATGGTATCAGCAGAAACAGGGAAGATCTCCTCAACTCCTGCTCTATAATGCAAAAACCTTAGCAGAAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGATTTATTACTGTCAACATTTTTCTGGAACTCCGTACACGTTCGGAGAGGGGACAAAATTGGAAATAACC SEQ ID No:9
  • Amino acid sequence of heavy chain variable region EVMLVESGGGLVKPGGSLKLSCAVSGFTFRNYAMSWVRQSPEKRLEWVAEISNGGRYIYYPDTVTGRFTISRDNAKNTLYLEMSSLRSEDTAMYYCVRGGEKTSGYVWFAYWGEGTLVTVG SEQ ID No:17
  • amino acid sequence of CDR1 in the variable region of the heavy chain is:
  • amino acid sequence of CDR2 in the variable region of the heavy chain is:
  • amino acid sequence of CDR3 in the variable region of the heavy chain is:
  • amino acid sequence of FR1 in the heavy chain variable region is:
  • amino acid sequence of FR2 of the heavy chain variable region is:
  • amino acid sequence of FR3 of the heavy chain variable region is:
  • amino acid sequence of FR4 of the heavy chain variable region is:
  • amino acid sequence of CDR1 in the light chain variable region is:
  • amino acid sequence of CDR2 of the light chain variable region is:
  • amino acid sequence of CDR3 in the light chain variable region is:
  • amino acid sequence of FR1 of the light chain variable region is:
  • amino acid sequence of FR2 of the light chain variable region is:
  • amino acid sequence of FR3 of the light chain variable region is:
  • amino acid sequence of FR4 of the light chain variable region is:
  • the heavy chain variable region gene is 363 bp in length, encoding 121 amino acid residues.
  • the heavy chain variable region nucleotide sequence is shown in SEQ ID NO: 1
  • the heavy chain variable region amino acid sequence is shown in SEQ ID NO: 17
  • the heavy chain CDR1 amino acid sequence is shown in SEQ ID No: 18
  • the heavy chain The amino acid sequence of CDR 2 is shown in SEQ ID No: 19, and the amino acid sequence of heavy chain CDR3 is shown in SEQ ID No: 20.
  • the light chain variable region gene sequence is 321 bp in length, encoding 107 amino acid residues.
  • the light chain variable region nucleotide sequence is shown in SEQ ID NO: 9
  • the light chain variable region amino acid sequence is shown in SEQ ID NO: 25
  • the light chain CDR 1 amino acid sequence is shown in SEQ ID No: 26
  • the light chain CDR 2 The amino acid sequence is shown in SEQ ID No: 27, and the light chain CDR 3 amino acid sequence is shown in SEQ ID No: 28.
  • the present invention provides a preparation method, identification and application of a monoclonal antibody against human fibroblast growth factor receptor 4.
  • the monoclonal antibody has the advantages of high sensitivity and good specificity, and is suitable for different detection methods.
  • the monoclonal antibody provided by the present invention can be widely used in Western Different methods such as blot, ELISA, flowCytometry to detect human fibroblast growth factor receptor 4 provide a basis for studying the function of human fibroblast growth factor receptor 4.
  • Figure 1 The application of monoclonal antibodies in flow cytometry.
  • mice with high antibody titer >1:100000 were selected for the fourth immunization, and the antigen protein was injected into the abdominal cavity. , Each mouse was injected with 100 ⁇ g.
  • the mice were killed and their spleen cells were fused with SP2/0 cells, and stable hybridoma cells were obtained by culturing in HAT medium.
  • FGFR4 antibody-secreting hybridoma cells were screened by ELISA method, and subcloned by limiting dilution method.
  • Monoclonal hybridoma cell lines that can secrete FGFR4 antibody were screened by ELISA method. The ELISA method was used to screen and obtain FGFR4 antibody-secreting monoclonal hybridoma cell lines. kind.
  • the ascites was affinity purified by Protein G (Protein G Sepharose 4 Fast Flow, GE Healthcare) to obtain the purified FGFR4 antibody protein, and the antibody concentration was measured by the BCA method. Run SDS-PAGE of the purified antibody (loading volume: 5.4 ⁇ g), and stain with Coomassie brilliant blue.
  • the monoclonal antibody hybridoma cells in the logarithmic growth phase were harvested, TRIZOL lysed for RNA extraction, cDNA was obtained after reverse transcription, and the heavy and light chain variable regions were amplified and obtained, and the non-functional VK gene was removed and cloned into pMD18 -T vector, sequencing, using IMGT/V-QUEST database for sequencing results comparison and further analysis.
  • the light chain variable region nucleotide sequence GACATCCAGATGACTCAGACTCCAGCCTCCCTATCTGCATCTGTGGAAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACAGTTATTTAGCATGGTATCAGCAGAAACAGGGAAGATCTCCTCAACTCCTGCTCTATAATGCAAAAACCTTAGCAGAAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGATTTATTACTGTCAACATTTTTCTGGAACTCCGTACACGTTCGGAGAGGGGACAAAATTGGAAATAACC SEQ ID No:9
  • Amino acid sequence of heavy chain variable region EVMLVESGGGLVKPGGSLKLSCAVSGFTFRNYAMSWVRQSPEKRLEWVAEISNGGRYIYYPDTVTGRFTISRDNAKNTLYLEMSSLRSEDTAMYYCVRGGEKTSGYVWFAYWGEGTLVTVG SEQ ID No:17
  • amino acid sequence of CDR1 in the variable region of the heavy chain is:
  • amino acid sequence of CDR2 in the variable region of the heavy chain is:
  • amino acid sequence of CDR3 in the variable region of the heavy chain is:
  • amino acid sequence of FR1 in the heavy chain variable region is:
  • amino acid sequence of FR2 of the heavy chain variable region is:
  • amino acid sequence of FR3 of the heavy chain variable region is:
  • amino acid sequence of FR4 of the heavy chain variable region is:
  • amino acid sequence of CDR1 in the light chain variable region is:
  • amino acid sequence of CDR2 of the light chain variable region is:
  • amino acid sequence of CDR3 in the light chain variable region is:
  • amino acid sequence of FR1 of the light chain variable region is:
  • amino acid sequence of FR2 of the light chain variable region is:
  • amino acid sequence of FR3 of the light chain variable region is:
  • amino acid sequence of FR4 of the light chain variable region is:
  • the heavy chain variable region gene is 363 bp in length, encoding 121 amino acid residues.
  • the heavy chain variable region nucleotide sequence is shown in SEQ ID NO: 1
  • the heavy chain variable region amino acid sequence is shown in SEQ ID NO: 17
  • the heavy chain CDR1 amino acid sequence is shown in SEQ ID No: 18
  • the heavy chain The amino acid sequence of CDR 2 is shown in SEQ ID No: 19, and the amino acid sequence of heavy chain CDR3 is shown in SEQ ID No: 20.
  • the light chain variable region gene sequence is 321 bp in length, encoding 107 amino acid residues.
  • the light chain variable region nucleotide sequence is shown in SEQ ID NO: 9
  • the light chain variable region amino acid sequence is shown in SEQ ID NO: 25
  • the light chain CDR1 amino acid sequence is shown in SEQ ID No: 26
  • the light chain CDR 2 The amino acid sequence is shown in SEQ ID No: 27, and the light chain CDR3 amino acid sequence is shown in SEQ ID No: 28.
  • the 6G12F5 antibody can bind to the FGFR4 protein on the surface of HepG2 cells, and is used in flow cytometry detection, and the detection sensitivity is better than the commercial positive control, and the fluorescence intensity shifts more and greater.
  • each well of the microtiter plate was coated with 100 ng FGFR4 protein and coated overnight at 4°C.
  • the 6G12F5 antibody was added at a concentration of 1 ⁇ g/ml, 100 ⁇ l per well, and incubated at 37°C for 1h. Wash the plate 5 times with PBST, add 1:5000 diluted HRP-goat anti-mouse IgG antibody, and incubate at 37°C for 45 min. Wash the plate 5 times with PBST, add 100 ⁇ l TMB to each well, and react for 3 min at room temperature in the dark. Add 50 ⁇ l stop solution to each well to stop, and measure the OD450 with a microplate reader. As shown in Figure 2, 6G12F5 can be used for ELISA detection applications.
  • 6G12F5 can be used for Western blot detection applications.

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Abstract

一种抗成纤维细胞生长因子受体4抗体及其制备方法和用途。涉及具体的抗成纤维细胞生长因子受体4抗体或其抗原结合片段，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。还涉及一种核苷酸序列，其特征在于：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

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 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（fibroblast growth 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%。
 技术问题
为了克服上述问题，本发明提供一种抗FGFR4的单克隆抗体6G12F5，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。
 技术解决方案
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: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。
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：17所示的重链可变区，如SEQ ID No:25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。
重链可变区核苷酸序列：
GAGGTGATGCTTGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGTCTCTGGATTCACTTTCAGGAACTATGCCATGTCTTGGGTTCGCCAGTCTCCAGAGAAGAGGCTGGAGTGGGTCGCAGAAATTAGTAATGGTGGTAGATATATCTACTATCCAGACACTGTGACGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGGAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGTAAGGGGGGGGGAAAAGACCTCGGGCTACGTCTGGTTTGCTTACTGGGGCGAAGGAACTTTAGTCACAGTCGGT              SEQ ID No:1
重链可变区CDR1核苷酸序列:
GGATTCACTTTCAGGAACTATGCC         SEQ ID No:2
重链可变区CDR2核苷酸序列
ATTAGTAATGGTGGTAGATATATC                  SEQ ID No:3
重链可变区CDR3核苷酸序列
GTAAGGGGGGGGGAAAAGACCTCGGGCTACGTCTGGTTTGCTTAC         SEQ ID No:4
重链可变区FR1核苷酸序列
GAGGTGATGCTTGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGTCTCT           SEQ ID No:5
重链可变区FR2核苷酸序列
ATGTCTTGGGTTCGCCAGTCTCCAGAGAAGAGGCTGGAGTGGGTCGCAGAA         SEQ ID No:6
重链可变区FR3核苷酸序列
TACTATCCAGACACTGTGACGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGGAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGT       SEQ ID No:7
重链可变区FR4核苷酸序列
TGGGGCGAAGGAACTTTAGTCACAGTCGGT        SEQ ID No:8
轻链可变区核苷酸序列：GACATCCAGATGACTCAGACTCCAGCCTCCCTATCTGCATCTGTGGAAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACAGTTATTTAGCATGGTATCAGCAGAAACAGGGAAGATCTCCTCAACTCCTGCTCTATAATGCAAAAACCTTAGCAGAAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGATTTATTACTGTCAACATTTTTCTGGAACTCCGTACACGTTCGGAGAGGGGACAAAATTGGAAATAACC              SEQ ID No:9
轻链可变区CDR1核苷酸序列
GAGAATATTTACAGTTAT   SEQ ID No:10
轻链可变区CDR2核苷酸序列
AATGCAAAA       SEQ ID No:11
轻链可变区CDR3核苷酸序列
CAACATTTTTCTGGAACTCCGTACACG   SEQ ID No:12
轻链可变区FR1核苷酸序列
GACATCCAGATGACTCAGACTCCAGCCTCCCTATCTGCATCTGTGGAAGAAACTGTCACCATCACATGTCGAGCAAGT       SEQ ID No:13
轻链可变区FR2核苷酸序列
TTAGCATGGTATCAGCAGAAACAGGGAAGATCTCCTCAACTCCTGCTCTAT      SEQ ID No:14
轻链可变区FR3核苷酸序列
ACCTTAGCAGAAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGATTTATTACTGT       SEQ ID No:15
轻链可变区FR4核苷酸序列
TTCGGAGAGGGGACAAAATTGGAAATAACC SEQ ID No:16
重链可变区氨基酸序列：EVMLVESGGGLVKPGGSLKLSCAVSGFTFRNYAMSWVRQSPEKRLEWVAEISNGGRYIYYPDTVTGRFTISRDNAKNTLYLEMSSLRSEDTAMYYCVRGGEKTSGYVWFAYWGEGTLVTVG       SEQ ID No:17
其中，重链可变区CDR1氨基酸序列为：
 GFTFRNYA  SEQ ID No: 18
重链可变区CDR2氨基酸序列为：
ISNGGRYI      SEQ ID No:19
重链可变区CDR3氨基酸序列为：
VRGGEKTSGYVWFAY  SEQ ID No:20
重链可变区FR1氨基酸序列为：
EVMLVESGGGLVKPGGSLKLSCAVS   SEQ ID No:21
重链可变区FR2氨基酸序列为：
MSWVRQSPEKRLEWVAE   SEQ ID No:22
重链可变区FR3氨基酸序列为：
YYPDTVTGRFTISRDNAKNTLYLEMSSLRSEDTAMYYC    SEQ ID No:23
重链可变区FR4氨基酸序列为：
WGEGTLVTVG     SEQ ID No:24
轻链可变区氨基酸序列：
DIQMTQTPASLSASVEETVTITCRASENIYSYLAWYQQKQGRSPQLLLYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGIYYCQHFSGTPYTFGEGTKLEIT       SEQ ID No:25
其中，轻链可变区CDR1氨基酸序列为：
ENIYSY   SEQ ID No:26
轻链可变区CDR2氨基酸序列为：
NAK       SEQ ID No:27
轻链可变区CDR3氨基酸序列为：
QHFSGTPYT  SEQ ID No:28
轻链可变区FR1氨基酸序列为：
DIQMTQTPASLSASVEETVTITCRAS  SEQ ID No:29
轻链可变区FR2氨基酸序列为：
LAWYQQKQGRSPQLLLY     SEQ ID No:30
轻链可变区FR3氨基酸序列为：
TLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGIYYC    SEQ ID No:31
轻链可变区FR4氨基酸序列为：
FGEGTKLEIT  SEQ ID No:32
重链可变区基因全长363bp，编码氨基酸残基121个。重链可变区核苷酸序列如SEQ ID NO:1所示，重链可变区氨基酸序列如SEQ ID NO:17所示，重链CDR1氨基酸序列如SEQ ID No: 18所示，重链CDR 2氨基酸序列如SEQ ID No: 19所示，重链CDR3氨基酸序列如SEQ ID No: 20所示。
轻链可变区基因序列全长321bp，编码氨基酸残基107个。轻链可变区核苷酸序列如SEQ ID NO: 9所示，轻链可变区氨基酸序列如SEQ ID NO: 25，轻链CDR 1氨基酸序列如SEQ ID No: 26所示，轻链CDR 2氨基酸序列如SEQ ID No: 27所示，轻链CDR 3氨基酸序列如SEQ ID No: 28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单克隆抗体在流式细胞检测中的应用。
图2单克隆抗体在ELISA检测中的应用。
图3单克隆抗体在Western blot检测中的应用。
 本发明的实施方式
为了使本发明的上述目的、特征和优点能够更加明显易懂，下面结合附图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1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100μg。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1~5×10 5个细胞（0.5ml），注射杂交瘤细胞~11天后处死小鼠，获得腹水。3000 rpm，4℃离心10 min，去除沉淀，用10倍体积1×PB溶液稀释腹水，混匀后过0.45 μm滤膜。通过Protein G （Protein G Sepharose 4 Fast 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5.4 μg），考马斯亮蓝染色。
实施例2 FGFR4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。
重链可变区核苷酸序列：
GAGGTGATGCTTGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGTCTCTGGATTCACTTTCAGGAACTATGCCATGTCTTGGGTTCGCCAGTCTCCAGAGAAGAGGCTGGAGTGGGTCGCAGAAATTAGTAATGGTGGTAGATATATCTACTATCCAGACACTGTGACGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGGAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGTAAGGGGGGGGGAAAAGACCTCGGGCTACGTCTGGTTTGCTTACTGGGGCGAAGGAACTTTAGTCACAGTCGGT              SEQ ID No:1
重链可变区CDR1核苷酸序列:
GGATTCACTTTCAGGAACTATGCC         SEQ ID No:2
重链可变区CDR2核苷酸序列
ATTAGTAATGGTGGTAGATATATC                  SEQ ID No:3
重链可变区CDR3核苷酸序列
GTAAGGGGGGGGGAAAAGACCTCGGGCTACGTCTGGTTTGCTTAC         SEQ ID No:4
重链可变区FR1核苷酸序列
GAGGTGATGCTTGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGTCTCT           SEQ ID No:5
重链可变区FR2核苷酸序列
ATGTCTTGGGTTCGCCAGTCTCCAGAGAAGAGGCTGGAGTGGGTCGCAGAA         SEQ ID No:6
重链可变区FR3核苷酸序列
TACTATCCAGACACTGTGACGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGGAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGT       SEQ ID No:7
重链可变区FR4核苷酸序列
TGGGGCGAAGGAACTTTAGTCACAGTCGGT        SEQ ID No:8
轻链可变区核苷酸序列：GACATCCAGATGACTCAGACTCCAGCCTCCCTATCTGCATCTGTGGAAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACAGTTATTTAGCATGGTATCAGCAGAAACAGGGAAGATCTCCTCAACTCCTGCTCTATAATGCAAAAACCTTAGCAGAAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGATTTATTACTGTCAACATTTTTCTGGAACTCCGTACACGTTCGGAGAGGGGACAAAATTGGAAATAACC              SEQ ID No:9
轻链可变区CDR1核苷酸序列
GAGAATATTTACAGTTAT   SEQ ID No:10
轻链可变区CDR2核苷酸序列
AATGCAAAA       SEQ ID No:11
轻链可变区CDR3核苷酸序列
CAACATTTTTCTGGAACTCCGTACACG   SEQ ID No:12
轻链可变区FR1核苷酸序列
GACATCCAGATGACTCAGACTCCAGCCTCCCTATCTGCATCTGTGGAAGAAACTGTCACCATCACATGTCGAGCAAGT       SEQ ID No:13
轻链可变区FR2核苷酸序列
TTAGCATGGTATCAGCAGAAACAGGGAAGATCTCCTCAACTCCTGCTCTAT      SEQ ID No:14
轻链可变区FR3核苷酸序列
ACCTTAGCAGAAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGATTTATTACTGT       SEQ ID No:15
轻链可变区FR4核苷酸序列
TTCGGAGAGGGGACAAAATTGGAAATAACC SEQ ID No:16
重链可变区氨基酸序列：EVMLVESGGGLVKPGGSLKLSCAVSGFTFRNYAMSWVRQSPEKRLEWVAEISNGGRYIYYPDTVTGRFTISRDNAKNTLYLEMSSLRSEDTAMYYCVRGGEKTSGYVWFAYWGEGTLVTVG       SEQ ID No:17
其中，重链可变区CDR1氨基酸序列为：
 GFTFRNYA  SEQ ID No: 18
重链可变区CDR2氨基酸序列为：
ISNGGRYI      SEQ ID No:19
重链可变区CDR3氨基酸序列为：
VRGGEKTSGYVWFAY  SEQ ID No:20
重链可变区FR1氨基酸序列为：
EVMLVESGGGLVKPGGSLKLSCAVS   SEQ ID No:21
重链可变区FR2氨基酸序列为：
MSWVRQSPEKRLEWVAE   SEQ ID No:22
重链可变区FR3氨基酸序列为：
YYPDTVTGRFTISRDNAKNTLYLEMSSLRSEDTAMYYC    SEQ ID No:23
重链可变区FR4氨基酸序列为：
WGEGTLVTVG     SEQ ID No:24
轻链可变区氨基酸序列：
DIQMTQTPASLSASVEETVTITCRASENIYSYLAWYQQKQGRSPQLLLYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGIYYCQHFSGTPYTFGEGTKLEIT       SEQ ID No:25
其中，轻链可变区CDR1氨基酸序列为：
ENIYSY   SEQ ID No:26
轻链可变区CDR2氨基酸序列为：
NAK       SEQ ID No:27
轻链可变区CDR3氨基酸序列为：
QHFSGTPYT  SEQ ID No:28
轻链可变区FR1氨基酸序列为：
DIQMTQTPASLSASVEETVTITCRAS  SEQ ID No:29
轻链可变区FR2氨基酸序列为：
LAWYQQKQGRSPQLLLY     SEQ ID No:30
轻链可变区FR3氨基酸序列为：
TLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGIYYC    SEQ ID No:31
轻链可变区FR4氨基酸序列为：
FGEGTKLEIT  SEQ ID No:32
重链可变区基因全长363bp，编码氨基酸残基121个。重链可变区核苷酸序列如SEQ ID NO:1所示，重链可变区氨基酸序列如SEQ ID NO:17所示，重链CDR1氨基酸序列如SEQ ID No: 18所示，重链CDR 2氨基酸序列如SEQ ID No: 19所示，重链CDR3氨基酸序列如SEQ ID No: 20所示。
轻链可变区基因序列全长321bp，编码氨基酸残基107个。轻链可变区核苷酸序列如SEQ ID NO:9所示，轻链可变区氨基酸序列如SEQ ID NO:25，轻链CDR1氨基酸序列如SEQ ID No: 26所示，轻链CDR 2氨基酸序列如SEQ ID No: 27所示，轻链CDR3氨基酸序列如SEQ ID No: 28所示。
实施例3 FGFR4单克隆抗体的亲和力测定
使用10 μg/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6G12F5抗体（566.7nM、283.3 nM、141.7 nM、70.8 nM、35.4 nM、17.7 nM、8.84 nM）作为流动相，使用分子间相互作用仪（OCTET K2，PALL life science）进行亲和力检测，程序设定为：Baseline 240 s，Loading 360 s，Baseline2 180 s，Association 480 s，Dissociation 480 s，使用AHC (Anti-hIgG Fc Capture)传感器。结果如下表所示，6G12F5抗体与FGFR4的亲和力<1.0E-12（M）。
Conc. (nM) Response KD (M) KD Error kon(1/Ms) kon Error kdis(1/s) RMax RMax Error kobs(1/s) Req Req/Rmax (%) Full X^2 Full R^2
125 0.7546 <1.0E-12 5.09E-11 9.96E+04 7.16E+02 <1.0E-07 0.7723 0.0016 1.24E-03 0.7723 100 0.9528 0.9925
62.5 0.6361 <1.0E-12 5.09E-11 9.96E+04 7.16E+02 <1.0E-07 0.772 0.0029 6.22E-03 0.772 100 0.9528 0.9925
31.3 0.5173 <1.0E-12 5.09E-11 9.96E+04 7.16E+02 <1.0E-07 0.8765 0.0049 3.11E-03 0.8765 100 0.9528 0.9925
15.6 0.364 <1.0E-12 5.09E-11 9.96E+04 7.16E+02 <1.0E-07 0.9985 0.0069 1.55E-03 0.9985 100 0.9528 0.9925
实施例4 FGFR4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3×10 5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：
Figure dest_path_image001
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100 μl DMEM培养液+1μl PE goat anti-mouse lgG(minimal x-reactivity) antibody（biolegend,405307），冰上孵育20 min。
4) 各加入1ml PBS+5% FBS，500 g离心5 min，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 CytoFLEX CM）检测。
如图1所示，6G12F5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，荧光强度偏移更多更大。
实施例5 FGFR4单克隆抗体的ELISA检测应用
酶标板每孔包被100 ng FGFR4蛋白，4℃包被过夜，经封闭洗板后，加入的6G12F5抗体，浓度为1μg/ml，每孔100 μl，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45 min。PBST洗板5次，每孔加入100 μl TMB，室温避光反应3 min，每孔加入50 μl终止液终止，酶标仪测OD450。如图2所示，6G12F5能用于ELISA检测应用。实施例6 FGFR4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6 min，跑SDS-PAGE，每孔上样20 μl，~500 ng蛋白/孔，400 mA转膜1 h，5%脱脂奶粉室温封闭1 h，加入6G12F5抗体（3 ml 5%脱脂奶粉中加入~10 μg抗体）4 ℃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3所示，6G12F5能用于Western blot检测应用。

Claims (9)

  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，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。
  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，其具有如SEQ ID No：17所示的重链可变区，如SEQ ID No:25所示的轻链可变区。
  3. 一种核苷酸序列，其特征在于：其编码如权利要求1或2所述的抗人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，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。
  6. 一种试剂盒，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，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作为检测试剂的用途，优选地，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。
  9. 权利要求1或2所述的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。
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