CN112608907B

Movatterモバイル変換

Info

Publication number: CN112608907B
Application number: CN202011507629.7A
Authority: CN
Inventors: 孟忠吉; 贺昱霖; 曾少波
Original assignee: Shiyan Taihe Hospital
Current assignee: Shiyan Taihe Hospital
Priority date: 2020-12-18
Filing date: 2020-12-18
Publication date: 2023-01-17
Anticipated expiration: 2040-12-18
Also published as: CN112608907A

Abstract

The invention discloses a monoclonal antibody which is produced by GPC3 full-length protein antigen immunity and can be specifically combined with glypican 3 (GPC 3) by a whole-gene synthesis method, a hybridoma cell strain for producing the monoclonal antibody and application thereof. The monoclonal antibody disclosed by the invention has high specificity and high sensitivity, can detect trace expression GPC3 protein, can be used as a reference index for clinical early diagnosis of liver cancer patients, and can be widely applied to clinical detection and treatment.

Description

Translated fromChinese

磷脂酰肌醇蛋白聚糖3单克隆抗体，杂交瘤细胞株和应用Glypican 3 monoclonal antibody, hybridoma cell line and application

技术领域：Technical field:

本发明属于杂交瘤细胞株领域，具体地说涉及一种磷脂酰肌醇蛋白聚糖 3(GPC3)单克隆抗体，杂交瘤细胞株制备和单克隆抗体的应用。The invention belongs to the field of hybridoma cell lines, in particular to a Glypican 3 (GPC3) monoclonal antibody, the preparation of hybridoma cell lines and the application of the monoclonal antibody.

背景技术：Background technique:

原发性肝癌(HCC)是全球常见恶性肿瘤之一，全世界每年超过78万人死于 HCC，每年新发病例84万，我国HCC的发病率和病死率占全球的55％以上，且呈逐年上升趋势。对于早期的HCC患者，主要采用早期肝切除术，这是目前 HCC治疗最有效的根治性手段。然而HCC起病隐匿，进展较快，很多患者在发现肿瘤时已经处于中晚期，丧失手术切除机会，而放射治疗、局部消融治疗、肝动脉化疗栓塞等治疗方法疗效有限，因此早期诊断和早期治疗是提高HCC患者生存率的关键。甲胎蛋白(AFP)是目前临床常用的肝癌特异性肿瘤标记物，然而，由于其灵敏度(39-64％)与特异性(76-91％)均有限，在部分慢性肝病和肝硬化患者，血清AFP水平也会升高，肝损伤和肝脏再生也能够增加非HCC患者血清AFP水平；近年来，异常凝血酶原(DCP，也称PIVKA-II)已被国内外指南列为肝癌检测极其重要的指标，但其特异性和敏感性仍然不足，而且在慢性肝炎和维生素K缺乏症也会轻度升高。因此寻找新的肝癌特异性标记物显得尤为重要。Primary liver cancer (HCC) is one of the common malignant tumors in the world. More than 780,000 people die from HCC every year in the world, and 840,000 new cases occur every year. rising trend year by year. For early HCC patients, early liver resection is mainly used, which is currently the most effective radical treatment for HCC. However, the onset of HCC is hidden and the progress is fast. Many patients are already in the middle and late stage when the tumor is discovered, and they lose the chance of surgical resection. The therapeutic effects of radiotherapy, local ablation therapy, and hepatic arterial chemoembolization are limited. Therefore, early diagnosis and early treatment It is the key to improve the survival rate of HCC patients. Alpha-fetoprotein (AFP) is currently a clinically commonly used specific tumor marker for liver cancer. However, due to its limited sensitivity (39-64%) and specificity (76-91%), in some patients with chronic liver disease and cirrhosis, Serum AFP levels will also increase, and liver injury and liver regeneration can also increase serum AFP levels in non-HCC patients; in recent years, abnormal prothrombin (DCP, also known as PIVKA-II) has been listed as extremely important for liver cancer detection by domestic and foreign guidelines index, but its specificity and sensitivity are still insufficient, and it will be slightly elevated in chronic hepatitis and vitamin K deficiency. Therefore, it is particularly important to find new specific markers for liver cancer.

磷脂酰肌醇蛋白聚糖3(GPC3)是硫酸乙酰肝素糖蛋白家族的一员，GPC3基因位于人类X染色体的26位，编码了580个氨基酸，分子量为66kD。研究显示，GPC3主要在滋养层和多种胚胎组织表达，在成人组织几乎不表达，当组织发生恶性转化时，GPC3 mRNA和蛋白就会再次表达，尤其在肝癌组织中高表达，因此被看作新的肝癌标记物，可以作为HCC临床早期诊断的参考指标。目前，还有研究发现，GPC3可能成为新的HCC免疫治疗靶点，能够对肿瘤靶细胞进行特异性杀伤。Glypican 3 (GPC3) is a member of the heparan sulfate glycoprotein family. The GPC3 gene is located at position 26 of the human X chromosome, encoding 580 amino acids with a molecular weight of 66 kD. Studies have shown that GPC3 is mainly expressed in trophoblasts and various embryonic tissues, and is almost not expressed in adult tissues. When malignant transformation occurs in tissues, GPC3 mRNA and protein will be expressed again, especially in liver cancer tissues, so it is regarded as a new Liver cancer markers can be used as reference indicators for early clinical diagnosis of HCC. At present, other studies have found that GPC3 may become a new target for HCC immunotherapy, which can specifically kill tumor target cells.

目前，市场销售的GPC3抗体多数为进口产品，价格昂贵，更重要的是的特异性和敏感性较低，限制了其在临床诊断方面以及靶向治疗方面的应用。目前市场销售的GPC3抗体价格昂贵且在实验中用量大，如Abcam公司的重组GPC3抗体 (货号ab207080)，免疫荧光检测使用浓度为1.136ug/ml，Western blot检测使用浓度为0.568ug/ml,100ul价格高达4853元。正因为如些，难以大规模应用。At present, most of the GPC3 antibodies sold in the market are imported products, which are expensive, and more importantly, their specificity and sensitivity are low, which limits their application in clinical diagnosis and targeted therapy. The GPC3 antibodies currently on the market are expensive and are used in large quantities in experiments, such as Abcam’s recombinant GPC3 antibody (product number ab207080), the concentration used for immunofluorescence detection is 1.136ug/ml, and the concentration used for Western blot detection is 0.568ug/ml, 100ul The price is as high as 4853 yuan. Because of this, it is difficult to apply on a large scale.

中国专利201210086014.0与201210086009.X公布了GPC3单克隆抗体杂交瘤细胞株8G6与7D11及其制备方法和应用，其中进行免疫荧光实验抗体需进行 1:10稀释，说明抗体用量大灵敏度不足。中国专利201510097025.2是用GPC3 人工半抗原作为免疫原制备GPC3单克隆抗体的，中国专利201110033378.8是用 GPC3-C端蛋白作为免疫原制备GPC3单克隆抗体的，中国专利201410767938.6 是用GPC3蛋白片段作为免疫原制备GPC3单克隆抗体的。上述专利的GPC3选取抗原不同，敏感性参差不齐，灵敏度差，重复性差。Chinese patents 201210086014.0 and 201210086009.X disclose the GPC3 monoclonal antibody hybridoma cell lines 8G6 and 7D11 and their preparation methods and applications. The antibodies need to be diluted 1:10 for immunofluorescence experiments, which shows that the sensitivity of the large amount of antibodies is insufficient. Chinese patent 201510097025.2 uses GPC3 artificial hapten as immunogen to prepare GPC3 monoclonal antibody, Chinese patent 201110033378.8 uses GPC3-C-terminal protein as immunogen to prepare GPC3 monoclonal antibody, Chinese patent 201410767938.6 uses GPC3 protein fragment as immunogen Preparation of GPC3 monoclonal antibody. The GPC3 of the above-mentioned patents has different antigens selected, the sensitivity is uneven, the sensitivity is poor, and the repeatability is poor.

发明技术内容：Invention technical content:

本发明的一个目的是提供高度灵敏性的单克隆抗体，该抗体由采用密码子优化及全基因合成的方法获得GPC3全长蛋白抗原免疫产生，可特异性地与磷脂酰肌醇蛋白聚糖3(GPC3)结合。An object of the present invention is to provide a highly sensitive monoclonal antibody, which is produced by immunizing the full-length protein antigen of GPC3 by adopting codon optimization and whole gene synthesis, and can specifically bind to Glypican 3 (GPC3) binding.

本发明的另一个目的是提供产生的单克隆抗体的杂交瘤细胞株，此细胞株可分泌抗GPC3蛋白的单克隆抗体，此抗体高度灵敏，重复性好。Another object of the present invention is to provide a hybridoma cell line producing a monoclonal antibody. This cell line can secrete a monoclonal antibody against GPC3 protein. The antibody is highly sensitive and has good reproducibility.

本发明的第三个目的是制备分泌GPC3抗体的杂交瘤细胞株的方法。The third object of the present invention is a method for preparing a hybridoma cell line secreting GPC3 antibody.

本发明的第四个目的是制备抗GPC3的单克隆抗体的方法。The fourth object of the present invention is a method for preparing an anti-GPC3 monoclonal antibody.

本发明的再一个目的是抗GPC3的单克隆抗体的应用。Another object of the present invention is the application of anti-GPC3 monoclonal antibody.

本发明公开了一株由核苷酸序列为SEQ SEQ ID NO:1表达的重组GPC3蛋白免疫产生的小鼠杂交瘤细胞株GPC3-T，该细胞株保藏在中国典型培养物保藏中心，保藏号为CCTCCNO:C2020109。本发明公开的杂交瘤细胞株是抗GPC3 蛋白的杂交瘤细胞株，本发明公开的作为免疫源使用的GPC3蛋白是由核苷酸序列为SEQ ID NO:1表达产生的，是采用密码子优化及全基因合成的方法获得 GPC3全长蛋白抗原，将其作为免疫原制备GPC3单克隆抗体，杂交瘤细胞是通过重组GPC3蛋白免疫小鼠脾细胞，再由免疫的小鼠脾细胞与骨髓瘤细胞融合获得。The invention discloses a mouse hybridoma cell line GPC3-T produced by immunization with a recombinant GPC3 protein expressed with a nucleotide sequence of SEQ ID NO: 1. The cell line is preserved in the China Center for Type Culture Collection, and the preservation number is for CCTCCNO:C2020109. The hybridoma cell strain disclosed in the present invention is a hybridoma cell strain resistant to GPC3 protein, and the GPC3 protein disclosed in the present invention used as an immune source is produced by expressing the nucleotide sequence of SEQ ID NO: 1, and adopts codon optimization and the whole gene synthesis method to obtain the GPC3 full-length protein antigen, and use it as an immunogen to prepare GPC3 monoclonal antibody. The hybridoma cells are immunized with mouse splenocytes by recombinant GPC3 protein, and then the immunized mouse splenocytes and myeloma cells are Fusion obtained.

本发明公开了在真核生物中表达制备纯化GPC3蛋白的方法。本发明的 GPC3全长蛋白抗原是采用密码子优化及全基因合成的方法获得GPC3全长蛋白抗原，将其作为免疫原制备GPC3单克隆抗体。GPC3蛋白具体的制备过程如下:The invention discloses a method for expressing and preparing purified GPC3 protein in eukaryotes. The GPC3 full-length protein antigen of the present invention is obtained by adopting codon optimization and whole gene synthesis methods to obtain the GPC3 full-length protein antigen, which is used as an immunogen to prepare a GPC3 monoclonal antibody. The specific preparation process of GPC3 protein is as follows:

1.引物设计与合成1. Primer design and synthesis

根据基因GPC3序列及重叠PCR原理设计引物，序列见表1，引物序列特征为：编号为单号的引物与GPC3基因序列(5'->3')方向一致，编号为双号的引物与GPC3基因序列(5'->3')反向互补，另外相邻编号的序列之间存在17bp重叠。第一条序列的前10bp为保护碱基。最后一条序列的后10bp为保护碱基。Primers were designed according to the gene GPC3 sequence and the principle of overlapping PCR. See Table 1 for the sequence. The gene sequence (5'->3') is reverse complementary, and there is a 17bp overlap between adjacent numbered sequences. The first 10bp of the first sequence is the protection base. The last 10 bp of the last sequence is the protection base.

表1引物列表Table 1 Primer list

待引物合成后用去离子水稀释至50pmol/μl，适当离心混合均匀。After primer synthesis, dilute to 50 pmol/μl with deionized water, and centrifuge properly to mix well.

2.基因扩增2. Gene Amplification

在冰上按表2配制反应体系，混匀后放入PCR仪，44条引物的相邻引物之间有17bp反向互补，每条引物中间有21bp左右未互补序列，44条引物涵盖了 GPC3基因的全长。将44条引物混合后经过退火处理(55℃，1min)，相邻引物的17bp序列互补配对。中间未配对的21bp左右序列，在高保真酶的作用下序列被补齐，从而构成完整的双链GPC3基因的全长序列。在PCR仪上经过25个循环扩增，GPC3基因得到扩增，PCR扩增程序见表3。Prepare the reaction system on ice according to Table 2, mix it and put it into the PCR instrument. There are 17 bp reverse complementary between the adjacent primers of the 44 primers, and there are about 21 bp uncomplementary sequences in the middle of each primer. The 44 primers cover GPC3 the full length of the gene. After the 44 primers were mixed, they were annealed (55°C, 1 min), and the 17bp sequences of adjacent primers were complementary paired. The unpaired sequence of about 21 bp in the middle is completed under the action of high-fidelity enzymes to form the full-length sequence of the complete double-stranded GPC3 gene. After 25 cycles of amplification on the PCR instrument, the GPC3 gene was amplified. The PCR amplification program is shown in Table 3.

表2 PCR扩增反应体系Table 2 PCR amplification reaction system

表3 PCR扩增反应条件Table 3 PCR amplification reaction conditions

3.扩增片段的电泳检测以及回收3. Electrophoretic detection and recovery of amplified fragments

PCR扩增后利用1.5％琼脂糖电泳30min，然后比照DNA marker大小，在紫外灯下回收目的条带，然后利用胶回收试剂盒回收目的基因GPC3条带。After PCR amplification, use 1.5% agarose electrophoresis for 30 minutes, then compare the size of the DNA marker, recover the target band under ultraviolet light, and then use the gel recovery kit to recover the target gene GPC3 band.

4.载体与目的基因的酶切4. Enzyme digestion of vector and target gene

表4酶切体系Table 4 enzyme digestion system

将回收的GPC3基因片段和pATX2载体分别按上述表4准备反应体系，然后在37℃酶切1-2h；将酶切产物利用1.5％的琼脂糖电泳检测并利用胶回收试剂盒回收酶切后的基因片段和载体片段。Prepare the reaction system for the recovered GPC3 gene fragment and pATX2 vector according to the above Table 4, and then digest at 37°C for 1-2 hours; use 1.5% agarose electrophoresis to detect the digested product and use the gel recovery kit to recover after digestion gene fragments and vector fragments.

5.载体与目的基因的连接：5. Connection of vector and target gene:

将酶切后的载体片段与GPC3基因片段按如下体系配制反应体系表5，并放于16℃连接1h。The digested vector fragment and the GPC3 gene fragment were prepared according to the following system to prepare the reaction system Table 5, and placed at 16°C for 1 hour for ligation.

表5连接体系Table 5 connection system

6.转化6. Conversion

将要经T4连接酶连接后的产品加入到装有TOP10感受态细胞的管中，50 μl感受态细胞需要25ng DNA，体积应不超过感受态细胞的5％，轻轻旋转几次混匀内容物，冰浴30min。Add the product to be ligated by T4 ligase into the tube containing TOP10 competent cells. 50 μl of competent cells requires 25ng DNA, and the volume should not exceed 5% of the competent cells. Gently rotate several times to mix the contents , ice bath for 30min.

将离心管混合物放入加温至42℃的循环水中，热激90s，不要摇动管。快速将管转移到冰浴中，使细胞冷却1～2min。Put the centrifuge tube mixture into circulating water warmed to 42°C, heat shock for 90s, do not shake the tube. Quickly transfer the tube to an ice bath and allow the cells to cool for 1-2 min.

每管加入200μl SOC液体培养基，用水浴将培养基加温至37℃，然后将管转移到设置为37℃的摇床上，220rpm培养45min，使细胞复苏并表达质粒编码的抗性标记基因。Add 200 μl of SOC liquid medium to each tube, warm the medium to 37 °C with a water bath, then transfer the tube to a shaker set at 37 °C, and incubate at 220 rpm for 45 min to allow the cells to recover and express the plasmid-encoded resistance marker gene.

将适当体积(每个90mm平板达200μl)已转化的感受态细胞转移到含有相应抗生素的LB培养基上。倒置平板，于37℃培养箱中培养12～16小时。An appropriate volume (up to 200 μl per 90 mm plate) of transformed competent cells was transferred to LB medium containing corresponding antibiotics. Invert the plate and incubate in a 37°C incubator for 12-16 hours.

7.菌落PCR验证7. Colony PCR verification

待平板上长出菌落，随机挑取若干个菌落，进行菌落PCR验证，检测转化子。After colonies grow on the plate, several colonies are randomly selected for colony PCR verification to detect transformants.

8.测序验证8. Sequencing Verification

阳性克隆送至测序公司测序验证，同时平行样品进行酶切电泳验证。Positive clones were sent to the sequencing company for sequencing verification, and parallel samples were verified by enzyme digestion and electrophoresis.

9.转染用质粒扩增与提取：利用无内毒素质粒提取试剂盒，提取质粒，此时含有GPC3基因片段的质粒命名为GPC3-pATX2。9. Amplification and extraction of plasmids for transfection: use an endotoxin-free plasmid extraction kit to extract plasmids. At this time, the plasmids containing the GPC3 gene fragments are named GPC3-pATX2.

质粒构建过程如附图1所示。The plasmid construction process is shown in Figure 1.

10.用GPC3-pATX2质粒转染HEK293细胞，转染后第6天收集细胞和培养基。10. HEK293 cells were transfected with GPC3-pATX2 plasmid, and the cells and culture medium were collected on the 6th day after transfection.

11.通过His标签进行纯化，用pH7.5的PBS进行平衡，然后用PBS pH 7.5， 30mM咪唑基，50mM咪唑基洗涤，最后用PBS pH 7.5，200mM咪唑基，400mM 咪唑基洗脱，通过SDS-PAGE定性和定量，并进行咪唑去除和浓缩，最终纯化得到的GPC3蛋白，核苷酸序列为SEQ IDNO:1。11. Purified by His tag, equilibrated with PBS pH 7.5, then washed with PBS pH 7.5, 30mM imidazolyl, 50mM imidazolyl, and finally eluted with PBS pH 7.5, 200mM imidazolyl, 400mM imidazolyl, by SDS -PAGE qualitative and quantitative, and carry out imidazole removal and concentration, and finally purify the obtained GPC3 protein, the nucleotide sequence is SEQ ID NO:1.

SEQ ID NO:1：SEQ ID NO: 1:

GAATTCATGGCCGGCACAGTCAGAACAGCTTGTCTGGTGGTGGCCATGCTGCTGTCACTGGACTTTCCAGG ACAGGCCCAACCTCCTCCTCCACCTCCTGATGCCACATGTCATCAAGTGCGGAGCTTTTTCCAGAGACTGCAGCCCGGCCTGAAATGGGTGCCAGAAACTCCTGTGCCTGGCAGCGACCTGCAAGTGTGCCTTCCTAAGGGCC CTACCTGCTGCAGCCGGAAGATGGAAGAGAAGTACCAGCTGACCGCCAGGCTGAACATGGAACAGCTGCT GCAGAGCGCCTCTATGGAACTGAAGTTCCTGATCATCCAGAACGCCGCCGTGTTCCAAGAGGCCTTCGAAAT CGTCGTGCGGCACGCCAAGAACTACACCAACGCCATGTTCAAGAACAACTACCCCAGCCTGACACCTCAGG CCTTTGAGTTCGTGGGCGAGTTCTTCACCGACGTGTCCCTGTACATCCTGGGCAGCGACATCAACGTGGAC GACATGGTCAACGAGCTGTTCGACTCTCTGTTCCCCGTGATCTACACCCAGCTGATGAACCCCGGCCTGCCT GATTCTGCCCTGGACATCAATGAGTGCCTGAGAGGCGCTAGACGGGACCTGAAGGTGTTCGGCAACTTCCC CAAGCTGATCATGACCCAGGTGTCCAAGTCTCTGCAAGTGACCCGGATCTTCCTGCAGGCCCTGAACCTGG GCATCGAAGTGATCAACACCACCGACCACCTGAAGTTCAGCAAGGACTGCGGCCGGATGCTGACCAGAATG TGGTACTGCAGCTACTGCCAGGGCCTGATGATGGTCAAGCCTTGCGGCGGCTACTGCAACGTTGTGATGCA GGGATGTATGGCCGGCGTGGTGGAAATCGACAAGTATTGGAGAGAGTACATCCTCAGCCTGGAAGAACTG GTCAACGGCATGTACCGGATCTACGACATGGAAAACGTGCTGCTGGGCCTGTTCAGCACCATCCACGACAG CATCCAGTACGTGCAGAAGAACGCCGGCAAGCTGACCACCACCATCGGAAAACTGTGTGCCCACAGCCAG CAGCGGCAGTACAGAAGCGCCTACTATCCCGAGGACCTGTTCATCGACAAGAAAGTGCTGAAGGTGGCCCA CGTGGAACACGAGGAAACCCTGAGCAGCAGAAGAAGAGAGCTGATCCAGAAGCTGAAGTCCTTTATCAGC TTCTACAGCGCCCTGCCTGGCTACATCTGCTCTCATTCTCCCGTGGCCGAGAACGACACCCTGTGCTGGAATG GCCAAGAGCTGGTGGAACGGTACAGCCAGAAAGCCGCCAGAAACGGCATGAAGAACCAGTTCAACCTGC ACGAGCTGAAGATGAAGGGCCCCGAGCCTGTGGTGTCCCAGATCATCGATAAGCTGAAGCACATCAACCAGCTGCTGAGGACCATGAGCATGCCCAAGGGCAGAGTGCTGGACAAGAACCTGGACGAGGAAGGCTTCGAG AGCGGCGATTGCGGAGATGACGAGGATGAGTGTATCGGCGGCAGCGGCGACGGCATGATCAAAGTGAAG AATCAGCTGCGGTTCCTGGCCGAGCTGGCCTACGATCTGGATGTGGATGATGCCCCTGGCAACTCCCAGCA GGCCACACCTAAGGACAACGAGATCTCCACCTTCCACAATCTGGGCAACGTGCACAGCCCTCTGAAGCTGC TGACCTCCATGGCTATCAGCGTCGTGTGCTTCTTCTTCCTGGTGCACGGCTCCCACCATCACCATCATCATAAG CTTGAATTCATGGCCGGCACAGTCAGAACAGCTTGTCTGGTGGTGGCCATGCTGCTGTCACTGGACTTTCCAGG ACAGGCCCAACCTCCTCCTCCACCTCCTGATGCCACATGTCATCAAGTGCGGAGCTTTTTCCAGAGACTGCAGCCCGGCCTGAAATGGGTGCCAGAAACTCCTGTGCCTGGCAGCGACCTGCAAGTGTGCCTTCCTAAGGGCC CTACCTGCTGCAGCCGGAAGATGGAAGAGAAGTACCAGCTGACCGCCAGGCTGAACATGGAACAGCTGCT GCAGAGCGCCTCTATGGAACTGAAGTTCCTGATCATCCAGAACGCCGCCGTGTTCCAAGAGGCCTTCGAAAT CGTCGTGCGGCACGCCAAGAACTACACCAACGCCATGTTCAAGAACAACTACCCCAGCCTGACACCTCAGG CCTTTGAGTTCGTGGGCGAGTTCTTCACCGACGTGTCCCTGTACATCCTGGGCAGCGACATCAACGTGGAC GACATGGTCAACGAGCTGTTCGACTCTCTGTTCCCCGTGATCTACACCCAGCTGATGAACCCCGGCCTGCCT GATTCTGCCCTGGACATCAATGAGTGCCTGAGAGGCGCTAGACGGGACCTGAAGGTGTTCGGCAACTTCCC CAAGCTGATCATGACCCAGGTGTCCAAGTCTCTGCAAGTGACCCGGATCTTCCTGCAGGCCCTGAACCTGG GCATCGAAGTGATCAACACCACCGACCACCTGAAGTTCAGCAAGGACTGCGGCCGGATGCTGACCAGAATG TGGTACTGCAGCTACTGCCAGGGCCTGATGATGGTCAAGCCTTGCGGCGGCTACTGCAACGTTGTGATGCA GGGATGTATGGCCGGCGTGGTGGAAATCGACAAGTATTGGAGAGAGTACATCCTCAGCCTGGAAGAACTG GTCAACGGCATGTACCGGATCTACGACATGGAAAACGTGCTGCTGGGCCTGTTCAGCACCATCC ACGACAG CATCCAGTACGTGCAGAAGAACGCCGGCAAGCTGACCACCACCATCGGAAAACTGTGTGCCCACAGCCAG CAGCGGCAGTACAGAAGCGCCTACTATCCCGAGGACCTGTTCATCGACAAGAAAGTGCTGAAGGTGGCCCA CGTGGAACACGAGGAAACCCTGAGCAGCAGAAGAAGAGAGCTGATCCAGAAGCTGAAGTCCTTTATCAGC TTCTACAGCGCCCTGCCTGGCTACATCTGCTCTCATTCTCCCGTGGCCGAGAACGACACCCTGTGCTGGAATG GCCAAGAGCTGGTGGAACGGTACAGCCAGAAAGCCGCCAGAAACGGCATGAAGAACCAGTTCAACCTGC ACGAGCTGAAGATGAAGGGCCCCGAGCCTGTGGTGTCCCAGATCATCGATAAGCTGAAGCACATCAACCAGCTGCTGAGGACCATGAGCATGCCCAAGGGCAGAGTGCTGGACAAGAACCTGGACGAGGAAGGCTTCGAG AGCGGCGATTGCGGAGATGACGAGGATGAGTGTATCGGCGGCAGCGGCGACGGCATGATCAAAGTGAAG AATCAGCTGCGGTTCCTGGCCGAGCTGGCCTACGATCTGGATGTGGATGATGCCCCTGGCAACTCCCAGCA GGCCACACCTAAGGACAACGAGATCTCCACCTTCCACAATCTGGGCAACGTGCACAGCCCTCTGAAGCTGC TGACCTCCATGGCTATCAGCGTCGTGTGCTTCTTCTTCCTGGTGCACGGCTCCCACCATCACCATCATCATAAG CTT

本发明还公开了一种抗GPC3的单克隆抗体，该抗体由杂交瘤细胞分泌所得，属于IgG亚型,能特异性地识别GPC3蛋白，并与GPC3阳性肝癌细胞系具有强反应性。本发明还公布了GPC3抗体的轻链可变区和重链可变区氨基酸序列，重链可变区的CDR1具有SEQ ID NO:46所示的氨基酸序列，CDR2具有SEQ ID NO:47所示的氨基酸序列，CDR3具有SEQ ID NO:48所示的氨基酸序列；轻链可变区CDR1具有SEQ ID NO:53所示的氨基酸序列，CDR2具有SEQID NO:54所示的氨基酸序列，CDR3具有SEQ ID NO:55所示的氨基酸序列。The invention also discloses an anti-GPC3 monoclonal antibody, which is secreted by hybridoma cells, belongs to IgG subtype, can specifically recognize GPC3 protein, and has strong reactivity with GPC3 positive liver cancer cell lines. The present invention also discloses the amino acid sequences of the light chain variable region and the heavy chain variable region of the GPC3 antibody, the CDR1 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:46, and the CDR2 has the amino acid sequence shown in SEQ ID NO:47 The amino acid sequence of CDR3 has the amino acid sequence shown in SEQ ID NO:48; The light chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO:53, and CDR2 has the amino acid sequence shown in SEQ ID NO:54, and CDR3 has the amino acid sequence shown in SEQ ID NO:54. Amino acid sequence shown in ID NO:55.

本发明公开了抗GPC3的单克隆抗体在制备诊断或治疗肿瘤的药物制剂的应用。The invention discloses the application of the anti-GPC3 monoclonal antibody in the preparation of pharmaceutical preparations for diagnosing or treating tumors.

本发明公开了制备小鼠杂交瘤细胞株的方法，其特征在于该方法包括：The invention discloses a method for preparing a mouse hybridoma cell line, which is characterized in that the method comprises:

A.通过密码子优化及基因合成蛋白全长的方法，构建GPC3质粒；A. Construct the GPC3 plasmid by codon optimization and gene synthesis of full-length protein;

B.用GPC3质粒转染HEK293细胞，转染后第6天收集细胞和培养基；B. HEK293 cells were transfected with GPC3 plasmid, and the cells and culture medium were collected on the 6th day after transfection;

C.进行表达纯化测试和发酵纯化获得目的GPC3蛋白；C. Perform expression purification test and fermentation purification to obtain the target GPC3 protein;

D.用GPC3抗原免疫小鼠，共免疫4-6次，检测第4次及第5次免疫后的抗血清效价；D. Immunize mice with GPC3 antigen, immunize 4-6 times in total, and detect the antiserum titer after the 4th and 5th immunization;

E.对抗血清效价达标的小鼠抗血清进行免疫荧光验证，选择免疫荧光验证阳性的小鼠，进入细胞融合，融合前冲击免疫，融合1-2次；ELISA的抗原蛋白筛选2-3次，将所有阳性细胞株上清收集并用免疫荧光等方法验证；E. Immunofluorescence verification of mouse antiserum whose antiserum titer reaches the standard, select mice with positive immunofluorescence verification, enter cell fusion, impact immunization before fusion, fusion 1-2 times; ELISA antigen protein screening 2-3 times , collect supernatants of all positive cell lines and verify by methods such as immunofluorescence;

F.取免疫小鼠脾细胞，再由免疫的小鼠脾细胞与骨髓瘤细胞融合筛选获得抗GPC3蛋白的杂交瘤细胞株；F. Take the splenocytes of the immunized mice, and then obtain the hybridoma cell line anti-GPC3 protein by fusion screening of the splenocytes of the immunized mice and myeloma cells;

G.将筛选的小鼠杂交瘤细胞株GPC3-T，送中国典型培养物保藏中心保藏，保藏号为CCTCC NO:C2020109。G. Send the screened mouse hybridoma cell line GPC3-T to the China Center for Type Culture Collection for preservation, and the preservation number is CCTCC NO: C2020109.

本发明还公开了抗GPC3的单克隆抗体的方法，其特征在于该方法包括：The present invention also discloses a method for anti-GPC3 monoclonal antibody, characterized in that the method comprises:

A.扩大培养杂交瘤细胞株GPC3-T；A. expanding the hybridoma cell line GPC3-T;

B.将杂交瘤细胞株GPC3-T注射入Balc/C小鼠腹腔，饲养小鼠，使小鼠产生腹水，收集腹水；B. Inject the hybridoma cell line GPC3-T into the peritoneal cavity of Balc/C mice, raise the mice, make the mice produce ascites, and collect the ascites;

C.过柱纯化得到抗GPC3蛋白的单克隆抗体。C. Column purification to obtain monoclonal antibody against GPC3 protein.

本发明还公开了抗GPC3的单克隆抗体，该抗体是鼠源抗体，抗体结构图见附图 2所示。The present invention also discloses an anti-GPC3 monoclonal antibody, which is a mouse antibody, and the structure diagram of the antibody is shown in Figure 2.

表1.重链CDR区氨基酸序列Table 1. Amino acid sequence of heavy chain CDR region

表2.重链FR区氨基酸序列Table 2. Amino acid sequence of heavy chain FR region

表3.轻链CDR区氨基酸序列Table 3. Amino acid sequence of light chain CDR region

表3.轻链FR区氨基酸序列Table 3. Amino acid sequence of light chain FR region

本发明的优点：与现有技术相比，本发明具有如下优点Advantages of the present invention: compared with the prior art, the present invention has the following advantages

1.高特异性。本发明中的GPC3单克隆抗体能够特异性结合肝癌患者血液和组织中的GPC3蛋白，可以作为HCC临床早期诊断的参考指标。与商品化GPC3 抗体相比，使用本发明中低浓度的GPC3单克隆抗体就能与Huh7细胞中的GPC3 蛋白结合产生绿色荧光，染色效果比使用其他品牌高浓度商品化抗体更优，而且本发明中的GPC3单克隆抗体不与不表达GPC3蛋白的细胞如A549细胞发生反应，特异性强；在蛋白印迹检测中，本发明中的GPC3单克隆抗体能够特异结合 GPC3阳性细胞蛋白，杂交条带单一，背景非常低，用1:8000浓度实验时，本发明中的GPC3单克隆抗体杂交条带亮度比其他品牌商品化抗体高30倍，且不与 GPC3阴性蛋白反应，具有高度的特异性。1. High specificity. The GPC3 monoclonal antibody of the present invention can specifically bind to the GPC3 protein in the blood and tissues of patients with liver cancer, and can be used as a reference index for early clinical diagnosis of HCC. Compared with the commercialized GPC3 antibody, the low concentration of GPC3 monoclonal antibody of the present invention can combine with the GPC3 protein in Huh7 cells to produce green fluorescence, and the staining effect is better than that of other brands of high-concentration commercialized antibodies, and the present invention The GPC3 monoclonal antibody in the present invention does not react with cells that do not express GPC3 protein, such as A549 cells, and has strong specificity; in Western blot detection, the GPC3 monoclonal antibody in the present invention can specifically bind to GPC3 positive cell proteins, and the hybridization band is single , the background is very low. When tested at a concentration of 1:8000, the hybridization band brightness of the GPC3 monoclonal antibody in the present invention is 30 times higher than that of commercial antibodies of other brands, and it does not react with GPC3-negative proteins and has a high degree of specificity.

2.高灵敏度。本发明中的GPC3单克隆抗体特异性强，且效价高于现有产品，在各种实验中使用浓度低，在免疫荧光法稀释度1:1600，比对照提高100倍；在蛋白印迹法稀释度1:8000比对照提高800倍，都可获得良好效果，本发明中的 GPC3单克隆抗体灵敏度高于现有产品，可检测到微量表达的GPC3蛋白。2. High sensitivity. The GPC3 monoclonal antibody in the present invention has strong specificity, and the titer is higher than that of existing products, and the concentration used in various experiments is low, and the dilution ratio of immunofluorescence method is 1:1600, which is 100 times higher than that of the control; The dilution ratio of 1:8000 is 800 times higher than that of the control, and good results can be obtained. The sensitivity of the GPC3 monoclonal antibody in the present invention is higher than that of existing products, and the GPC3 protein expressed in a small amount can be detected.

3.本发明中的GPC3单克隆抗体可以直接用于抗体药物或靶向GPC3的CAR-T 细胞治疗研究。3. The GPC3 monoclonal antibody in the present invention can be directly used in the research of antibody drug or CAR-T cell therapy targeting GPC3.

4.而本发明中的GPC3单克隆抗体，免疫荧光检测仅需0.4625ug/ml浓度即可，Western blot仅需0.0925ug/ml浓度。与Abcam公司的重组GPC3抗体(货号 ab207080)相比，在免疫荧光时用量节约60％，在Western blot时节约在可以减少使用量，节约成本83.7％，效果明显。4. For the GPC3 monoclonal antibody of the present invention, only a concentration of 0.4625ug/ml is required for immunofluorescence detection, and only a concentration of 0.0925ug/ml is required for Western blot. Compared with Abcam's recombinant GPC3 antibody (catalogue number ab207080), it saves 60% in immunofluorescence, saves in Western blot, and saves 83.7% in cost. The effect is obvious.

附图说明Description of drawings

图1是GPC3-pATX2质粒构建流程示意图Figure 1 is a schematic diagram of the GPC3-pATX2 plasmid construction process

图2是GPC3单克隆抗体结构示意图Figure 2 is a schematic diagram of the structure of the GPC3 monoclonal antibody

图3是GPC3蛋白SDS-PAGE电泳纯化图(MW:Molecular weight marker；IN:Input；FT:Flow through；W:Washes；E:Eluted fractions)Figure 3 is the SDS-PAGE electrophoresis purification diagram of GPC3 protein (MW: Molecular weight marker; IN: Input; FT: Flow through; W: Washes; E: Eluted fractions)

图4是用免疫荧光法比较本发明中GPC3单克隆抗体和某公司商品化GPC3抗体在表达GPC3蛋白的细胞系Huh7细胞中的检测效果Figure 4 is a comparison of the detection effect of the GPC3 monoclonal antibody of the present invention and a commercial GPC3 antibody of a certain company in the cell line Huh7 cells expressing GPC3 protein by immunofluorescence

图5是用免疫荧光法比较本发明中GPC3单克隆抗体和某公司商品化GPC3抗体在不表达GPC3蛋白的细胞系A549细胞中的检测效果Figure 5 is a comparison of the detection effect of the GPC3 monoclonal antibody of the present invention and a commercial GPC3 antibody of a company in the cell line A549 cells that do not express GPC3 protein by immunofluorescence

图6是用Western blot法比较本发明中GPC3单克隆抗体和某公司商品化GPC3 抗体在Huh7细胞蛋白、转染PCDNA3.1-GPC3质粒(GPC3过表达)和PCDNA3.1 空载质粒的A549细胞蛋白中的检测效果Figure 6 is a Western blot comparison of the GPC3 monoclonal antibody of the present invention and a company's commercial GPC3 antibody in Huh7 cell protein, A549 cells transfected with pDNA3.1-GPC3 plasmid (GPC3 overexpression) and pDNA3.1 empty plasmid Detection effect in protein

具体实施方式detailed description

下面结合具体的实施例来进一步阐述本发明。应当理解，这些实施例仅用于说明本发明，而不能限制本发明的保护范围。The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention, but not to limit the protection scope of the present invention.

实施例Example

实施例1磷脂酰肌醇蛋白聚糖3(GPC3)蛋白制备Embodiment 1 Glypican 3 (GPC3) protein preparation

1.1.引物设计与合成1.1. Primer design and synthesis

1.2.基因扩增1.2. Gene amplification

1.3.扩增片段的电泳检测以及回收1.3. Electrophoretic detection and recovery of amplified fragments

1.4.载体与目的基因的酶切1.4. Enzyme digestion of vector and target gene

1.5.载体与目的基因的连接：1.5. Connection of vector and target gene:

1.6.转化1.6. Conversion

将要经T4连接酶连接后的产品加入到装有TOP10感受态细胞的管中(50 μl感受态细胞需要25ng DNA)，体积应不超过感受态细胞的5％，轻轻旋转几次混匀内容物，冰浴30min。Add the product to be ligated by T4 ligase into the tube containing TOP10 competent cells (25ng DNA is required for 50 μl competent cells), the volume should not exceed 5% of the competent cells, gently swirl several times to mix the contents objects, ice-bathed for 30min.

1.7.菌落PCR验证1.7. Colony PCR verification

1.8.测序验证1.8. Sequencing verification

1.9.转染用质粒扩增与提取：利用无内毒素质粒提取试剂盒，提取质粒，此时含有GPC3基因片段的质粒命名为GPC3-pATX2。1.9. Amplification and extraction of the plasmid for transfection: the plasmid was extracted using an endotoxin-free plasmid extraction kit, and the plasmid containing the GPC3 gene fragment was named GPC3-pATX2.

质粒构建过程如图1。The plasmid construction process is shown in Figure 1.

1.10.用GPC3-pATX2质粒转染HEK293细胞，转染后第6天收集细胞和培养基。1.10. HEK293 cells were transfected with GPC3-pATX2 plasmid, and the cells and culture medium were collected on the 6th day after transfection.

1.11.通过His标签进行纯化，用pH7.5的PBS进行平衡，然后用PBS pH 7.5，30mM咪唑基，50mM咪唑基洗涤，最后用PBS pH 7.5，200mM咪唑基，400mM咪唑基洗脱，通过SDS-PAGE定性和定量，并进行咪唑去除和浓缩，最终纯化得到的GPC3蛋白。1.11. Purify by His tag, equilibrate with PBS pH 7.5, then wash with PBS pH 7.5, 30mM imidazolyl, 50mM imidazolyl, and finally elute with PBS pH 7.5, 200mM imidazolyl, 400mM imidazolyl, by SDS -PAGE for qualitative and quantitative analysis, followed by imidazole removal and concentration, and final purification of the resulting GPC3 protein.

1.12.用GPC3质粒转染HEK293细胞，转染后第6天收集细胞和培养基。1.12. HEK293 cells were transfected with GPC3 plasmid, and the cells and culture medium were collected on the 6th day after transfection.

1.13.通过His标签进行纯化，用pH7.5的PBS进行平衡，然后用PBS pH 7.5， 30mM咪唑基，50mM咪唑基洗涤，最后用PBS pH 7.5，200mM咪唑基，400mM 咪唑基洗脱，通过SDS-PAGE定性和定量，结果如图3A所示，将W2至E5合并，并进行咪唑去除和浓缩，最终纯化得到的GPC3蛋白如图3B所示。1.13. Purify by His tag, equilibrate with PBS pH 7.5, then wash with PBS pH 7.5, 30mM imidazolyl, 50mM imidazolyl, and finally elute with PBS pH 7.5, 200mM imidazolyl, 400mM imidazolyl, by SDS -PAGE qualitative and quantitative, the results are shown in Figure 3A, W2 to E5 were combined, imidazole was removed and concentrated, and the final purified GPC3 protein was shown in Figure 3B.

实施例2鼠抗人GPC3单克隆抗体的制备Example 2 Preparation of mouse anti-human GPC3 monoclonal antibody

2.1、以GPC3蛋白为免疫原免疫BaLb/c小鼠。2.1. BaLb/c mice were immunized with GPC3 protein as immunogen.

初次免疫，抗原50ug加福氏完全佐剂皮下多点注射；2周后第二次免疫，剂量同上，加福氏不完全佐剂；2周后第三次，剂量同上，加福氏不完全佐剂，5-7 天后采血测其效价，检测免疫效果(表1)；2周后加强免疫，剂量50-100μg为宜，不加佐剂，3天后，取脾融合。For the first immunization, the antigen 50ug was injected subcutaneously with complete Freund’s adjuvant in multiple points; 2 weeks later, the second immunization was given the same dose as above, with Freund’s incomplete adjuvant; for the third time after 2 weeks, the dose was the same as above, and Freund’s incomplete adjuvant was added. After 5-7 days, the blood was collected to measure its potency and detect the immune effect (Table 1); 2 weeks later, boost the immunization with a dose of 50-100 μg without adjuvant. After 3 days, the spleen was taken for fusion.

表4小鼠血清ELISA检测数据表Table 4 Mouse serum ELISA detection data table

编号Numbering10001000200020004000400080008000160001600032000320006400064000阴性对照negative control111.981.981.271.270.800.800.430.430.300.300.270.270.210.210.020.02222.252.251.641.641.141.140.600.600.370.370.260.260.130.130.030.03330.910.910.460.460.300.300.190.190.150.150.120.120.140.140.020.02441.561.560.850.850.440.440.240.240.180.180.100.100.070.070.020.02552.292.291.351.350.810.810.590.590.320.320.190.190.140.140.040.04

2.2、选择免疫反应最好的小鼠进行细胞融合，选择杂交瘤。2.2. Select the mice with the best immune response for cell fusion and select hybridomas.

2.2.1取对数生长的骨髓瘤细胞SP2/0，1000rpm离心5分钟，弃上清，用不完全培养液混悬细胞后计数，取所需的细胞数，用不完全培养液洗涤2次。2.2.1 Take the logarithmic growth myeloma cells SP2/0, centrifuge at 1000rpm for 5 minutes, discard the supernatant, suspend the cells with incomplete culture medium and count, get the required number of cells, and wash twice with incomplete culture medium .

2.2.2同时制备免疫脾细胞悬液，用不完全培养液洗涤2次。2.2.2 Prepare immune spleen cell suspension at the same time, wash with incomplete culture medium twice.

2.2.3将骨髓瘤细胞与脾细胞按1∶10或1∶5的比例混合在一起，在50ml塑料离心管内用不完全培养液洗1次，1200rpm，8分钟。2.2.3 Mix myeloma cells and splenocytes at a ratio of 1:10 or 1:5, wash once with incomplete culture medium in a 50ml plastic centrifuge tube, 1200rpm, 8 minutes.

2.2.4弃上清，用滴管吸净残留液体，以免影响PEG的浓度。2.2.4 Discard the supernatant, and suck up the residual liquid with a dropper, so as not to affect the concentration of PEG.

2.2.5轻轻弹击离心管底，使细胞沉淀略加松动。2.2.5 Gently flick the bottom of the centrifuge tube to loosen the cell pellet slightly.

2.2.6在室温下融合:2.2.6 Fusion at room temperature:

①30秒内加入预热的1ml 45％PEG含5％DMSO，边加边搅拌。① Add 1ml ofpreheated 45% PEG containing 5% DMSO within 30 seconds, and stir while adding.

②作用90秒钟，若冬天室温较低时可延长至120秒钟。②The effect is 90 seconds, if the room temperature is low in winter, it can be extended to 120 seconds.

③加预热的不完全培养液，终止PEG作用，每隔2分钟分别加入1ml，2ml，3ml， 4ml，5ml和10ml。③Add preheated incomplete culture medium to terminate the PEG effect, and add 1ml, 2ml, 3ml, 4ml, 5ml and 10ml every 2 minutes.

2.2.7离心，800rpm，6分钟。2.2.7 Centrifuge at 800 rpm for 6 minutes.

2.2.8弃上清，HAT选择培养液轻轻混悬，切记不能用力吹打，以免使融合在一起的细胞散开。2.2.8 Discard the supernatant, and gently suspend in the HAT selection culture medium. Remember not to blow hard, so as not to disperse the fused cells.

2.2.9将融合后细胞悬液加入96孔板，200μl/孔，37℃、5％CO2孵箱培养。2.2.9 Add the fused cell suspension to a 96-well plate, 200 μl/well, and culture in a 37°C, 5% CO2 incubator.

2.2.10间接ELISA进行克隆筛选。2.2.10 Indirect ELISA for clone screening.

2.3、杂交瘤的克隆化和冻存。2.3. Cloning and cryopreservation of hybridomas.

2.3.1挑选OD值最高的孔进行有限稀释法筛选亚克隆2.3.1 Select the well with the highest OD value to screen subclones by limiting dilution

①阳性孔细胞的计数，并调细胞数在1～5×10³/ml。① Count the cells in the positive wells and adjust the cell number to 1-5×10³ /ml.

②取130个细胞放入6.5ml HT培养液，即20个细胞/ml，100μl/孔加A、B、 C三排为每孔2个细胞。余下2.9ml细胞悬液补加2.9ml HT培养液,细胞数为10 个/ml，100μl/孔加D、E、F三排，为每孔1个细胞。余下2.2ml细胞悬液补加 2.2mlHT培养液，细胞数5个/ml，100μl/孔，加G、H两排，为每孔0.5个细胞。② Take 130 cells and put them into 6.5ml HT medium, that is, 20 cells/ml, add 100μl/well and add three rows of A, B, and C to make 2 cells per well. Add 2.9ml HT culture medium to the remaining 2.9ml cell suspension, the number of cells is 10/ml, add D, E, F three rows to 100μl/well, 1 cell per well. Add 2.2ml HT medium to the remaining 2.2ml cell suspension, the number of cells is 5/ml, 100μl/well, add two rows of G and H, 0.5 cells per well.

③第8-9天时，肉眼可见细胞克隆，及时进行抗体检测，通过间接ELISA进行阳性克隆筛选。③ On day 8-9, cell clones are visible to the naked eye, and antibody detection is performed in time, and positive clones are screened by indirect ELISA.

2.3.2杂交瘤细胞的冻存2.3.2 Cryopreservation of hybridoma cells

细胞冻存要求：冻存方法同其他细胞系的冻存方法一样，每支含1×10⁶以上，冻存细胞要定期复苏，检查细胞的活性和分泌抗体的稳定性。Requirements for cryopreservation of cells: The cryopreservation method is the same as that of other cell lines, each vial contains more than 1×10⁶ , and the frozen cells should be revived regularly to check the activity of the cells and the stability of the secreted antibodies.

(3)将一株稳定分泌抗人GPC3单克隆抗体的小鼠杂交瘤细胞株GPC3-T保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:C2020109。保藏日期是2020 年11月25日。中国典型培养物保藏中心位于湖北省武汉市武昌区武汉大学校内，电话：027-68752319，EMAIL：cctcc@whu.edu.cn.在本发明公开后，第三方研究者可依法从保藏机构获取此细胞用于再现本发明的技术内容。(3) A mouse hybridoma cell line GPC3-T stably secreting anti-human GPC3 monoclonal antibody was deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: C2020109. The deposit date is November 25, 2020. The Chinese Typical Culture Collection Center is located in Wuhan University, Wuchang District, Wuhan City, Hubei Province, Tel: 027-68752319, EMAIL:cctcc@whu.edu.cn . After the disclosure of the invention, third-party researchers can obtain it from the depository institution according to law. Cells are used to reproduce the technical content of the present invention.

2.4、腹水生产及纯化。2.4. Ascites production and purification.

2.4.1腹水的制备2.4.1 Preparation of ascites

先腹腔注射0.5mlPristane(降植烷)于BaLb/c鼠，1～2周后腹腔注射1×106个杂交瘤细胞，接种细胞7～10天后可产生腹水，注射器抽取腹水，可反复收集数次。2.4.2Protein A/G纯化抗体Inject 0.5ml Pristane (pristane) intraperitoneally into BaLb/c mice first, and then inject 1×106 hybridoma cells intraperitoneally after 1 to 2 weeks. Ascites can be produced 7 to 10 days after the cells are inoculated, and the ascites can be extracted with a syringe, which can be collected several times . 2.4.2 Protein A/G purified antibody

①预处理。使用前去掉Protein A或者Protein G resin的保存液体，然后把填料装进洗净的纯化柱，再用约10倍体积的PBS洗涤一至三次。① Pretreatment. Remove the preservation liquid of Protein A or Protein G resin before use, then put the filler into the cleaned purification column, and then wash with about 10 times the volume of PBS for one to three times.

②上腹水。将腹水先进行离心，5000g离心20min左右，离心完后，油脂等杂质漂浮在腹水上面，细胞等其它较重的成份则沉在底部，取中间的清澈的腹水加到装好的柱子里，然后封住柱子置于摇床上摇动，室温2h或4度过夜。② ascites. Centrifuge the ascites first at 5000g for about 20 minutes. After the centrifugation, impurities such as oils and fats float on the ascites, and other heavy components such as cells sink to the bottom. Take the clear ascites in the middle and add it to the installed column, and then Seal the column and place it on a shaker for 2 hours or overnight at room temperature.

③洗涤与洗脱。将腹水从亲和柱内流出。再用10倍柱体积的PBS洗柱3次，最后用洗脱液将抗体洗脱下即可。③ Washing and elution. Drain the ascites from the affinity column. Then wash the column 3 times with 10 times the column volume of PBS, and finally use the eluent to elute the antibody.

实施例3GPC3单克隆抗体在免疫荧光实验中的应用Application of embodiment 3GPC3 monoclonal antibody in immunofluorescence experiment

3.1、将Huh7细胞与A549细胞分别制成单细胞悬液，加入到铺有无菌玻片的培养皿中，放置于细胞培养箱中培养，细胞会贴壁到无菌玻片上。3.1. Make single-cell suspensions of Huh7 cells and A549 cells respectively, add them to a culture dish covered with sterile glass slides, place them in a cell incubator for culture, and the cells will adhere to the sterile glass slides.

3.2、取出玻片，用PBS轻轻冲洗3遍，避免损伤细胞。3.2. Take out the slide and gently rinse it with PBS 3 times to avoid damaging the cells.

3.3、将玻片置于玻片盒中，加入4％的多聚甲醛固定液固定10min，用PBS洗3 次，每次5min。3.3. Put the slides in a slide box, add 4% paraformaldehyde fixative solution to fix for 10 minutes, wash with PBS 3 times, 5 minutes each time.

3.4、在玻片上滴加0.3％Triton破膜15min，PBS洗3次，每次5min。3.4. Add 0.3% Triton dropwise on the slide to rupture the membrane for 15 minutes, wash with PBS 3 times, 5 minutes each time.

3.5、用山羊血清室温封闭10min，弃去。3.5. Block with goat serum for 10 minutes at room temperature, then discard.

3.6、滴加以1:1600比例稀释(使用浓度0.4625ug/ml)后的GPC3单克隆抗体，某公司商品化GPC3抗体使用比例为1:200(使用浓度1.136ug/ml)将玻片置于湿盒内，放入37℃温箱中，1h。3.6. Add the GPC3 monoclonal antibody diluted 1:1600 (used concentration 0.4625ug/ml) dropwise, and the commercialized GPC3 antibody of a company uses a ratio of 1:200 (used concentration 1.136ug/ml) and place the slide in a wet In the box, put it in a 37°C incubator for 1h.

3.7、取出玻片，用PBS洗3次，每次5min。3.7. Take out the slide and wash with PBS 3 times, 5min each time.

3.8、滴加二抗(山羊抗小鼠)，37℃避光孵育30min后，用PBS洗3次，每次5min。3.8. Add the secondary antibody (goat anti-mouse) dropwise, incubate at 37°C in the dark for 30 minutes, then wash with PBS 3 times, 5 minutes each time.

3.9、DAPI复染核，2-3min，PBS洗3次，每次5min。3.9. DAPI counterstained nuclei, 2-3min, washed 3 times with PBS, 5min each time.

3.10、封片，荧光显微镜下观察，拍照。3.10. Seal the slides, observe under a fluorescence microscope, and take pictures.

结果显示，经GPC3单克隆抗体和对照商品化GPC3抗体染色的Huh7细胞均能观察到绿色荧光，而且使用本发明中低浓度的GPC3单克隆抗体，染色效果比使用高浓度商品化抗体更优(图4)，证明本发明中的GPC3单克隆抗体灵敏度高，而且不表达GPC3蛋白的A549细胞经本发明中的GPC3单克隆抗体染色后，未观察到绿色荧光(图5)，证明GPC3单克隆抗体可以特异性识别天然的人GPC3 蛋白。而且与对照商品化GPC3抗体相比，在免疫荧光时用量节约60％，效果明显。The results show that green fluorescence can be observed in Huh7 cells stained with GPC3 monoclonal antibody and contrasting commercialized GPC3 antibody, and the staining effect is better than that of high-concentration commercialized antibody ( Fig. 4), prove that the GPC3 monoclonal antibody of the present invention has high sensitivity, and after the A549 cells that do not express GPC3 protein are stained by the GPC3 monoclonal antibody of the present invention, no green fluorescence is observed (Fig. 5), proving that the GPC3 monoclonal antibody The antibody can specifically recognize native human GPC3 protein. Moreover, compared with the control commercial GPC3 antibody, the amount used in immunofluorescence is saved by 60%, and the effect is obvious.

实施例4GPC3单克隆抗体在western blot中的应用Application of embodiment 4GPC3 monoclonal antibody in western blot

4.1、以PCDNA3.1-GPC3质粒(GPC3过表达质粒)和PCDNA3.1空载质粒(对照质粒)转染A549细胞，提取蛋白，检测GPC3单克隆抗体对原核表达GPC3蛋白的识别作用，提取Huh7细胞蛋白，检测GPC3单克隆抗体对真核表达GPC3蛋白的识别作用；用某公司商品化GPC3抗体作为对照。4.1. Transfect A549 cells with PCDNA3.1-GPC3 plasmid (GPC3 overexpression plasmid) and PCDNA3.1 empty plasmid (control plasmid), extract protein, detect the recognition effect of GPC3 monoclonal antibody on prokaryotic GPC3 protein, and extract Huh7 For cell protein, the recognition effect of GPC3 monoclonal antibody on eukaryotic GPC3 protein was detected; the commercial GPC3 antibody of a certain company was used as a control.

4.2、将上述提取的蛋白进行SDS-PAGE凝胶电泳，根据凝胶大小准备滤纸、 PVDF膜，并将其浸泡于转膜缓冲液中，将转移槽底座放在转膜液中，按照正极、海绵、滤纸、凝胶、PVDF膜、滤纸、海绵垫、负极的顺序安装，注意避免气泡产生，否则影响转膜效果，采用恒流350mA，转膜2h，转膜完毕取出膜。4.2. Perform SDS-PAGE gel electrophoresis on the above-mentioned extracted protein, prepare filter paper and PVDF membrane according to the size of the gel, soak them in the transfer buffer, place the base of the transfer tank in the transfer solution, follow the positive electrode, Sponge, filter paper, gel, PVDF membrane, filter paper, sponge pad, and negative electrode are installed in order. Pay attention to avoid air bubbles, otherwise it will affect the transfer effect. Use a constant current of 350mA, transfer the membrane for 2 hours, and take out the membrane after transfer.

4.3、封闭：将转膜完毕的PVDF膜放置于封闭液中，摇床上室温封闭1.5h。4.3. Sealing: place the transferred PVDF membrane in the blocking solution, and seal at room temperature for 1.5 hours on a shaker.

4.4、孵育一抗：弃去封闭液，将PVDF膜置于GPC3单克隆抗体溶液1:2000稀释(浓度0.37ug/ml)、1:8000稀释(浓度0.0925ug/ml)、某公司商品化GPC3抗体 1:1000稀释(浓度0.568ug/ml)中，摇床上4℃孵育，过夜。4.4. Incubate the primary antibody: Discard the blocking solution, place the PVDF membrane in the GPC3 monoclonal antibody solution 1:2000 dilution (concentration 0.37ug/ml), 1:8000 dilution (concentration 0.0925ug/ml), a commercial GPC3 Dilute the antibody 1:1000 (concentration 0.568ug/ml), and incubate overnight at 4°C on a shaker.

4.5、孵育二抗：将PVDF膜放置于辣根过氧化物酶(HRP)标记的抗小鼠二抗 (1:2000稀释)溶液中，摇床上室温孵育2h。4.5. Secondary antibody incubation: Place the PVDF membrane in horseradish peroxidase (HRP)-labeled anti-mouse secondary antibody (1:2000 dilution) solution, and incubate at room temperature on a shaker for 2 hours.

4.6、显影：按A液与B液1:1的比例配制显影液，将配制好的显影液滴加于PVDF 膜正面，室温作用2min，显影拍照。4.6. Developing: Prepare a developing solution according to the ratio of 1:1 between liquid A and liquid B, add the prepared developing solution dropwise on the front of the PVDF membrane, let it work for 2 minutes at room temperature, develop and take pictures.

结果显示(图6)，本发明中的GPC3单克隆抗体既可以识别人肝癌细胞株Huh7 表达的GPC3蛋白，也可以识别真核瞬时表达的GPC3蛋白，不识别不表达GPC3 的蛋白，说明本发明中的GPC3单克隆抗体特异性强，而且本发明中仅用低浓度的GPC3单克隆抗体杂交条带亮度就比高浓度商品化抗体高30倍，说明本发明中的GPC3单克隆抗体灵敏度高，在Westernblot时可以减少使用量，节约成本83.7％，效果明显。The results show (Figure 6), the GPC3 monoclonal antibody in the present invention can recognize the GPC3 protein expressed by the human liver cancer cell line Huh7, and can also recognize the GPC3 protein transiently expressed in eukaryotic cells, and does not recognize the protein that does not express GPC3, which illustrates the present invention The GPC3 monoclonal antibody in the present invention has strong specificity, and the brightness of the hybridization band with only a low concentration of the GPC3 monoclonal antibody in the present invention is 30 times higher than that of a high-concentration commercial antibody, indicating that the GPC3 monoclonal antibody in the present invention has high sensitivity. In Western blot, the usage can be reduced, and the cost can be saved by 83.7%. The effect is obvious.

实施例5GPC3单克隆抗体序列Embodiment 5GPC3 monoclonal antibody sequence

通过对GPC3杂交瘤细胞GPC3-T进行测序，获得GPC3抗体重链及轻链全长序列(FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4)。By sequencing the GPC3 hybridoma cell GPC3-T, the full-length sequence of the heavy chain and light chain of the GPC3 antibody (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4) was obtained.

序列表sequence listing

<110> 十堰市太和医院（湖北医药学院附属医院）<110> Shiyan Taihe Hospital (Affiliated Hospital of Hubei University of Medicine)

<120> 磷脂酰肌醇蛋白聚糖3单克隆抗体，杂交瘤细胞株和应用<120> Glypican 3 monoclonal antibody, hybridoma cell line and application

<140> 202011507629.7<140> 202011507629.7

<141> 2020-12-18<141> 2020-12-18

<160> 59<160> 59

<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0

<210> 1<210> 1

<211> 1776<211> 1776

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence (Artificial Sequence)

<400> 1<400> 1

gaattcatgg ccggcacagt cagaacagct tgtctggtgg tggccatgct gctgtcactg 60gaattcatgg ccggcacagt cagaacagct tgtctggtgg tggccatgct gctgtcactg 60

gactttccag gacaggccca acctcctcct ccacctcctg atgccacatg tcatcaagtg 120gactttccag gacaggccca acctcctcct ccacctcctg atgccacatg tcatcaagtg 120

cggagctttt tccagagact gcagcccggc ctgaaatggg tgccagaaac tcctgtgcct 180cggagctttt tccagagact gcagcccggc ctgaaatggg tgccagaaac tcctgtgcct 180

ggcagcgacc tgcaagtgtg ccttcctaag ggccctacct gctgcagccg gaagatggaa 240ggcagcgacc tgcaagtgtg ccttcctaag ggccctacct gctgcagccg gaagatggaa 240

gagaagtacc agctgaccgc caggctgaac atggaacagc tgctgcagag cgcctctatg 300gagaagtacc agctgaccgc caggctgaac atggaacagc tgctgcagag cgcctctatg 300

gaactgaagt tcctgatcat ccagaacgcc gccgtgttcc aagaggcctt cgaaatcgtc 360gaactgaagt tcctgatcat ccagaacgcc gccgtgttcc aagaggcctt cgaaatcgtc 360

gtgcggcacg ccaagaacta caccaacgcc atgttcaaga acaactaccc cagcctgaca 420gtgcggcacg ccaagaacta caccaacgcc atgttcaaga acaactaccc cagcctgaca 420

cctcaggcct ttgagttcgt gggcgagttc ttcaccgacg tgtccctgta catcctgggc 480cctcaggcct ttgagttcgt gggcgagttc ttcaccgacg tgtccctgta catcctgggc 480

agcgacatca acgtggacga catggtcaac gagctgttcg actctctgtt ccccgtgatc 540agcgacatca acgtggacga catggtcaac gagctgttcg actctctgtt ccccgtgatc 540

tacacccagc tgatgaaccc cggcctgcct gattctgccc tggacatcaa tgagtgcctg 600tacacccagc tgatgaaccc cggcctgcct gattctgccc tggacatcaa tgagtgcctg 600

agaggcgcta gacgggacct gaaggtgttc ggcaacttcc ccaagctgat catgacccag 660agaggcgcta gacgggacct gaaggtgttc ggcaacttcc ccaagctgat catgacccag 660

gtgtccaagt ctctgcaagt gacccggatc ttcctgcagg ccctgaacct gggcatcgaa 720gtgtccaagt ctctgcaagt gacccggatc ttcctgcagg ccctgaacct gggcatcgaa 720

gtgatcaaca ccaccgacca cctgaagttc agcaaggact gcggccggat gctgaccaga 780gtgatcaaca ccaccgacca cctgaagttc agcaaggact gcggccggat gctgaccaga 780

atgtggtact gcagctactg ccagggcctg atgatggtca agccttgcgg cggctactgc 840atgtggtact gcagctactg ccagggcctg atgatggtca agccttgcgg cggctactgc 840

aacgttgtga tgcagggatg tatggccggc gtggtggaaa tcgacaagta ttggagagag 900aacgttgtga tgcagggatg tatggccggc gtggtggaaa tcgacaagta ttggagagag 900

tacatcctca gcctggaaga actggtcaac ggcatgtacc ggatctacga catggaaaac 960tacatcctca gcctggaaga actggtcaac ggcatgtacc ggatctacga catggaaaac 960

gtgctgctgg gcctgttcag caccatccac gacagcatcc agtacgtgca gaagaacgcc 1020gtgctgctgg gcctgttcag caccatccac gacagcatcc agtacgtgca gaagaacgcc 1020

ggcaagctga ccaccaccat cggaaaactg tgtgcccaca gccagcagcg gcagtacaga 1080ggcaagctga ccaccaccat cggaaaactg tgtgcccaca gccagcagcg gcagtacaga 1080

agcgcctact atcccgagga cctgttcatc gacaagaaag tgctgaaggt ggcccacgtg 1140agcgcctact atcccgagga cctgttcatc gacaagaaag tgctgaaggt ggcccacgtg 1140

gaacacgagg aaaccctgag cagcagaaga agagagctga tccagaagct gaagtccttt 1200gaacacgagg aaaccctgag cagcagaaga agagagctga tccagaagct gaagtccttt 1200

atcagcttct acagcgccct gcctggctac atctgctctc attctcccgt ggccgagaac 1260atcagcttct acagcgccct gcctggctac atctgctctc attctcccgt ggccgagaac 1260

gacaccctgt gctggaatgg ccaagagctg gtggaacggt acagccagaa agccgccaga 1320gacaccctgt gctggaatgg ccaagagctg gtggaacggt acagccagaa agccgccaga 1320

aacggcatga agaaccagtt caacctgcac gagctgaaga tgaagggccc cgagcctgtg 1380aacggcatga agaaccagtt caacctgcac gagctgaaga tgaagggccc cgagcctgtg 1380

gtgtcccaga tcatcgataa gctgaagcac atcaaccagc tgctgaggac catgagcatg 1440gtgtcccaga tcatcgataa gctgaagcac atcaaccagc tgctgaggac catgagcatg 1440

cccaagggca gagtgctgga caagaacctg gacgaggaag gcttcgagag cggcgattgc 1500cccaagggca gagtgctgga caagaacctg gacgaggaag gcttcgagag cggcgattgc 1500

ggagatgacg aggatgagtg tatcggcggc agcggcgacg gcatgatcaa agtgaagaat 1560ggagatgacg aggatgagtg tatcggcggc agcggcgacg gcatgatcaa agtgaagaat 1560

cagctgcggt tcctggccga gctggcctac gatctggatg tggatgatgc ccctggcaac 1620cagctgcggt tcctggccga gctggcctac gatctggatg tggatgatgc ccctggcaac 1620

tcccagcagg ccacacctaa ggacaacgag atctccacct tccacaatct gggcaacgtg 1680tcccagcagg ccacacctaa ggacaacgag atctccacct tccacaatct gggcaacgtg 1680

cacagccctc tgaagctgct gacctccatg gctatcagcg tcgtgtgctt cttcttcctg 1740cacagccctc tgaagctgct gacctccatg gctatcagcg tcgtgtgctt cttcttcctg 1740

gtgcacggct cccaccatca ccatcatcat aagctt 1776gtgcacggct cccaccatca ccatcatcat aagctt 1776

<210> 2<210> 2

<211> 59<211> 59

<212> DNA<212> DNA

<400> 2<400> 2

ccaatccaat gaattcatgg ccggcacagt cagaacagct tgtctggtgg tggccatgc 59ccaatccaat gaattcatgg ccggcacagt cagaacagct tgtctggtgg tggccatgc 59

<210> 3<210> 3

<211> 55<211> 55

<212> DNA<212> DNA

<400> 3<400> 3

aggaggttgg gcctgtcctg gaaagtccag tgacagcagc atggccacca ccaga 55aggaggttgg gcctgtcctg gaaagtccag tgacagcagc atggccacca ccaga 55

<210> 4<210> 4

<211> 55<211> 55

<212> DNA<212> DNA

<400> 4<400> 4

gacaggccca acctcctcct ccacctcctg atgccacatg tcatcaagtg cggag 55gacaggccca acctcctcct ccaccctcctg atgccacatg tcatcaagtg cggag 55

<210> 5<210> 5

<211> 55<211> 55

<212> DNA<212> DNA

<400> 5<400> 5

gcacccattt caggccgggc tgcagtctct ggaaaaagct ccgcacttga tgaca 55gcaccattt caggccgggc tgcagtctct ggaaaaagct ccgcacttga tgaca 55

<210> 6<210> 6

<211> 55<211> 55

<212> DNA<212> DNA

<400> 6<400> 6

cggcctgaaa tgggtgccag aaactcctgt gcctggcagc gacctgcaag tgtgc 55cggcctgaaa tgggtgccag aaactcctgt gcctggcagc gacctgcaag tgtgc 55

<210> 7<210> 7

<211> 55<211> 55

<212> DNA<212> DNA

<400> 7<400> 7

tccatcttcc ggctgcagca ggtagggccc ttaggaaggc acacttgcag gtcgc 55tccatcttcc ggctgcagca ggtagggccc ttaggaaggc acacttgcag gtcgc 55

<210> 8<210> 8

<211> 55<211> 55

<212> DNA<212> DNA

<400> 8<400> 8

tgcagccgga agatggaaga gaagtaccag ctgaccgcca ggctgaacat ggaac 55tgcagccgga agatggaaga gaagtaccag ctgaccgcca ggctgaacat ggaac 55

<210> 9<210> 9

<211> 55<211> 55

<212> DNA<212> DNA

<400> 9<400> 9

caggaacttc agttccatag aggcgctctg cagcagctgt tccatgttca gcctg 55caggaacttc agttccatag aggcgctctg cagcagctgt tccatgttca gcctg 55

<210> 10<210> 10

<211> 55<211> 55

<212> DNA<212> DNA

<400> 10<400> 10

tggaactgaa gttcctgatc atccagaacg ccgccgtgtt ccaagaggcc ttcga 55tggaactgaa gttcctgatc atccagaacg ccgccgtgtt ccaagaggcc ttcga 55

<210> 11<210> 11

<211> 54<211> 54

<212> DNA<212> DNA

<400> 11<400> 11

tggcgttggt gtagttcttg gcgtgccgca cgacgatttc gaaggcctct tgga 54tggcgttggt gtagttcttg gcgtgccgca cgacgatttc gaaggcctct tgga 54

<210> 12<210> 12

<211> 55<211> 55

<212> DNA<212> DNA

<400> 12<400> 12

gaactacacc aacgccatgt tcaagaacaa ctaccccagc ctgacacctc aggcc 55gaactacacc aacgccatgt tcaagaacaa ctaccccagc ctgacacctc aggcc 55

<210> 13<210> 13

<211> 55<211> 55

<212> DNA<212> DNA

<400> 13<400> 13

agggacacgt cggtgaagaa ctcgcccacg aactcaaagg cctgaggtgt caggc 55agggacacgt cggtgaagaa ctcgcccacg aactcaaagg cctgaggtgt caggc 55

<210> 14<210> 14

<211> 55<211> 55

<212> DNA<212> DNA

<400> 14<400> 14

ttcaccgacg tgtccctgta catcctgggc agcgacatca acgtggacga catgg 55ttcaccgacg tgtccctgta catcctgggc agcgacatca acgtggacga catgg 55

<210> 15<210> 15

<211> 55<211> 55

<212> DNA<212> DNA

<400> 15<400> 15

gtagatcacg gggaacagag agtcgaacag ctcgttgacc atgtcgtcca cgttg 55gtagatcacg gggaacagag agtcgaacag ctcgttgacc atgtcgtcca cgttg 55

<210> 16<210> 16

<211> 55<211> 55

<212> DNA<212> DNA

<400> 16<400> 16

tgttccccgt gatctacacc cagctgatga accccggcct gcctgattct gccct 55tgttccccgt gatctacacc cagctgatga accccggcct gcctgattct gccct 55

<210> 17<210> 17

<211> 55<211> 55

<212> DNA<212> DNA

<400> 17<400> 17

ggtcccgtct agcgcctctc aggcactcat tgatgtccag ggcagaatca ggcag 55ggtcccgtct agcgcctctc aggcactcat tgatgtccag ggcagaatca ggcag 55

<210> 18<210> 18

<211> 55<211> 55

<212> DNA<212> DNA

<400> 18<400> 18

aggcgctaga cgggacctga aggtgttcgg caacttcccc aagctgatca tgacc 55aggcgctaga cgggacctga aggtgttcgg caacttcccc aagctgatca tgacc 55

<210> 19<210> 19

<211> 55<211> 55

<212> DNA<212> DNA

<400> 19<400> 19

aggaagatcc gggtcacttg cagagacttg gacacctggg tcatgatcag cttgg 55aggaagatcc gggtcacttg cagagacttg gacacctggg tcatgatcag cttgg 55

<210> 20<210> 20

<211> 58<211> 58

<212> DNA<212> DNA

<400> 20<400> 20

gtgacccgga tcttcctgca ggccctgaac ctgggcatcg aagtgatcaa caccaccg 58gtgacccgga tcttcctgca ggccctgaac ctgggcatcg aagtgatcaa caccaccg 58

<210> 21<210> 21

<211> 59<211> 59

<212> DNA<212> DNA

<400> 21<400> 21

tggtcagcat ccggccgcag tccttgctga acttcaggtg gtcggtggtg ttgatcact 59tggtcagcat ccggccgcag tccttgctga acttcaggtg gtcggtggtg ttgatcact 59

<210> 22<210> 22

<211> 59<211> 59

<212> DNA<212> DNA

<400> 22<400> 22

cggccggatg ctgaccagaa tgtggtactg cagctactgc cagggcctga tgatggtca 59cggccggatg ctgaccagaa tgtggtactg cagctactgc cagggcctga tgatggtca 59

<210> 23<210> 23

<211> 59<211> 59

<212> DNA<212> DNA

<400> 23<400> 23

tacatccctg catcacaacg ttgcagtagc cgccgcaagg cttgaccatc atcaggccc 59tacatccctg catcacaacg ttgcagtagc cgccgcaagg cttgaccatc atcaggccc 59

<210> 24<210> 24

<211> 59<211> 59

<212> DNA<212> DNA

<400> 24<400> 24

tgtgatgcag ggatgtatgg ccggcgtggt ggaaatcgac aagtattgga gagagtaca 59tgtgatgcag ggatgtatgg ccggcgtggt ggaaatcgac aagtattgga gagagtaca 59

<210> 25<210> 25

<211> 59<211> 59

<212> DNA<212> DNA

<400> 25<400> 25

agatccggta catgccgttg accagttctt ccaggctgag gatgtactct ctccaatac 59agatccggta catgccgttg accagttctt ccaggctgag gatgtactct ctccaatac 59

<210> 26<210> 26

<211> 59<211> 59

<212> DNA<212> DNA

<400> 26<400> 26

cggcatgtac cggatctacg acatggaaaa cgtgctgctg ggcctgttca gcaccatcc 59cggcatgtac cggatctacg acatggaaaa cgtgctgctg ggcctgttca gcaccatcc 59

<210> 27<210> 27

<211> 59<211> 59

<212> DNA<212> DNA

<400> 27<400> 27

tcagcttgcc ggcgttcttc tgcacgtact ggatgctgtc gtggatggtg ctgaacagg 59tcagcttgcc ggcgttcttc tgcacgtact ggatgctgtc gtggatggtg ctgaacagg 59

<210> 28<210> 28

<211> 59<211> 59

<212> DNA<212> DNA

<400> 28<400> 28

gaacgccggc aagctgacca ccaccatcgg aaaactgtgt gcccacagcc agcagcggc 59gaacgccggc aagctgacca ccaccatcgg aaaactgtgt gcccacagcc agcagcggc 59

<210> 29<210> 29

<211> 59<211> 59

<212> DNA<212> DNA

<400> 29<400> 29

tgtcgatgaa caggtcctcg ggatagtagg cgcttctgta ctgccgctgc tggctgtgg 59tgtcgatgaa caggtcctcg ggatagtagg cgcttctgta ctgccgctgc tggctgtgg 59

<210> 30<210> 30

<211> 59<211> 59

<212> DNA<212> DNA

<400> 30<400> 30

ggacctgttc atcgacaaga aagtgctgaa ggtggcccac gtggaacacg aggaaaccc 59ggacctgttc atcgacaaga aagtgctgaa ggtggcccac gtggaacacg aggaaaccc 59

<210> 31<210> 31

<211> 59<211> 59

<212> DNA<212> DNA

<400> 31<400> 31

aggacttcag cttctggatc agctctcttc ttctgctgct cagggtttcc tcgtgttcc 59aggacttcag cttctggatc agctctcttc ttctgctgct cagggtttcc tcgtgttcc 59

<210> 32<210> 32

<211> 59<211> 59

<212> DNA<212> DNA

<400> 32<400> 32

ccagaagctg aagtccttta tcagcttcta cagcgccctg cctggctaca tctgctctc 59ccagaagctg aagtccttta tcagcttcta cagcgccctg cctggctaca tctgctctc 59

<210> 33<210> 33

<211> 59<211> 59

<212> DNA<212> DNA

<400> 33<400> 33

ggccattcca gcacagggtg tcgttctcgg ccacgggaga atgagagcag atgtagcca 59ggccattcca gcacagggtg tcgttctcgg ccacgggaga atgagagcag atgtagcca 59

<210> 34<210> 34

<211> 59<211> 59

<212> DNA<212> DNA

<400> 34<400> 34

cctgtgctgg aatggccaag agctggtgga acggtacagc cagaaagccg ccagaaacg 59cctgtgctgg aatggccaag agctggtgga acggtacagc cagaaagccg ccagaaacg 59

<210> 35<210> 35

<211> 59<211> 59

<212> DNA<212> DNA

<400> 35<400> 35

ccttcatctt cagctcgtgc aggttgaact ggttcttcat gccgtttctg gcggctttc 59ccttcatctt cagctcgtgc aggttgaact ggttcttcat gccgtttctg gcggctttc 59

<210> 36<210> 36

<211> 59<211> 59

<212> DNA<212> DNA

<400> 36<400> 36

cgagctgaag atgaagggcc ccgagcctgt ggtgtcccag atcatcgata agctgaagc 59cgagctgaag atgaagggcc ccgagcctgt ggtgtcccag atcatcgata agctgaagc 59

<210> 37<210> 37

<211> 59<211> 59

<212> DNA<212> DNA

<400> 37<400> 37

tgcccttggg catgctcatg gtcctcagca gctggttgat gtgcttcagc ttatcgatg 59tgcccttggg catgctcatg gtcctcagca gctggttgat gtgcttcagc ttatcgatg 59

<210> 38<210> 38

<211> 59<211> 59

<212> DNA<212> DNA

<400> 38<400> 38

gagcatgccc aagggcagag tgctggacaa gaacctggac gaggaaggct tcgagagcg 59gagcatgccc aagggcagag tgctggacaa gaacctggac gaggaaggct tcgagagcg 59

<210> 39<210> 39

<211> 59<211> 59

<212> DNA<212> DNA

<400> 39<400> 39

cgctgccgcc gatacactca tcctcgtcat ctccgcaatc gccgctctcg aagccttcc 59cgctgccgcc gatacactca tcctcgtcat ctccgcaatc gccgctctcg aagccttcc 59

<210> 40<210> 40

<211> 59<211> 59

<212> DNA<212> DNA

<400> 40<400> 40

gtgtatcggc ggcagcggcg acggcatgat caaagtgaag aatcagctgc ggttcctgg 59gtgtatcggc ggcagcggcg acggcatgat caaagtgaag aatcagctgc ggttcctgg 59

<210> 41<210> 41

<211> 59<211> 59

<212> DNA<212> DNA

<400> 41<400> 41

tgccaggggc atcatccaca tccagatcgt aggccagctc ggccaggaac cgcagctga 59tgccaggggc atcatccaca tccagatcgt aggccagctc ggccaggaac cgcagctga 59

<210> 42<210> 42

<211> 59<211> 59

<212> DNA<212> DNA

<400> 42<400> 42

ggatgatgcc cctggcaact cccagcaggc cacacctaag gacaacgaga tctccacct 59ggatgatgcc cctggcaact cccagcaggc cacacctaag gacaacgaga tctccacct 59

<210> 43<210> 43

<211> 59<211> 59

<212> DNA<212> DNA

<400> 43<400> 43

tcagcagctt cagagggctg tgcacgttgc ccagattgtg gaaggtggag atctcgttg 59tcagcagctt cagagggctg tgcacgttgc ccagattgtg gaaggtggag atctcgttg 59

<210> 44<210> 44

<211> 59<211> 59

<212> DNA<212> DNA

<400> 44<400> 44

ccctctgaag ctgctgacct ccatggctat cagcgtcgtg tgcttcttct tcctggtgc 59ccctctgaag ctgctgacct ccatggctat cagcgtcgtg tgcttcttct tcctggtgc 59

<210> 45<210> 45

<211> 59<211> 59

<212> DNA<212> DNA

<400> 45<400> 45

ccaatccaat aagcttatga tgatggtgat ggtgggagcc gtgcaccagg aagaagaag 59ccaatccaat aagcttatga tgatggtgat ggtgggagcc gtgcaccagg aagaagaag 59

<210> 46<210> 46

<211> 8<211> 8

<212> PRT<212> PRT

<400> 46<400> 46

Gly Tyr Thr Phe Thr Ser Tyr TrpGly Tyr Thr Phe Thr Ser Tyr Trp

1 51 5

<210> 47<210> 47

<211> 8<211> 8

<212> PRT<212> PRT

<400> 47<400> 47

Ile Tyr Pro Ser Asp Ser Tyr ThrIle Tyr Pro Ser Asp Ser Tyr Thr

1 51 5

<210> 48<210> 48

<211> 15<211> 15

<212> PRT<212> PRT

<400> 48<400> 48

Thr Arg Thr Tyr Tyr Ser Ser Glu Arg Phe Tyr Ala Met Asp TyrThr Arg Thr Tyr Tyr Ser Ser Glu Arg Phe Tyr Ala Met Asp Tyr

1 5 10 151 5 10 15

<210> 49<210> 49

<211> 25<211> 25

<212> PRT<212> PRT

<400> 49<400> 49

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly AlaGln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 151 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala SerSer Val Lys Leu Ser Cys Lys Ala Ser

20 25 20 25

<210> 50<210> 50

<211> 17<211> 17

<212> PRT<212> PRT

<400> 50<400> 50

Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile GlyIle Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly

1 5 10 151 5 10 15

AsnAsn

<210> 51<210> 51

<211> 38<211> 38

<212> PRT<212> PRT

<400> 51<400> 51

Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp LysAsn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys

1 5 10 151 5 10 15

Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu AspSer Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp

20 25 30 20 25 30

Ser Ala Val Tyr Tyr CysSer Ala Val Tyr Tyr Cys

35 35

<210> 52<210> 52

<211> 11<211> 11

<212> PRT<212> PRT

<400> 52<400> 52

Trp Gly Gln Gly Thr Ser Val Thr Val Ser SerTrp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

1 5 101 5 10

<210> 53<210> 53

<211> 6<211> 6

<212> PRT<212> PRT

<400> 53<400> 53

Gln Gly Ile His Asn TyrGln Gly Ile His Asn Tyr

1 51 5

<210> 54<210> 54

<211> 3<211> 3

<212> PRT<212> PRT

<400> 54<400> 54

Tyr Thr SerTyr Thr Ser

11

<210> 55<210> 55

<211> 9<211> 9

<212> PRT<212> PRT

<400> 55<400> 55

Gln Gln Tyr Ser Leu Leu Pro Trp ThrGln Gln Tyr Ser Leu Leu Pro Trp Thr

1 51 5

<210> 56<210> 56

<211> 26<211> 26

<212> PRT<212> PRT

<400> 56<400> 56

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu GlyAsp Ile Gln Met Thr Gln Thr Thr Ser Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 151 5 10 15

Asp Arg Val Thr Ile Ser Cys Ser Ala SerAsp Arg Val Thr Ile Ser Cys Ser Ala Ser

20 25 20 25

<210> 57<210> 57

<211> 17<211> 17

<212> PRT<212> PRT

<400> 57<400> 57

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu IleLeu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

1 5 10 151 5 10 15

TyrTyr

<210> 58<210> 58

<211> 36<211> 36

<212> PRT<212> PRT

<400> 58<400> 58

Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser GlySer Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

1 5 10 151 5 10 15

Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro Glu Asp Ile AlaThr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro Glu Asp Ile Ala

20 25 30 20 25 30

Thr Tyr Tyr CysThr Tyr Tyr Cys

35 35

<210> 59<210> 59

<211> 10<211> 10

<212> PRT<212> PRT

<400> 59<400> 59

Phe Gly Gly Gly Thr Lys Leu Glu Ile LysPhe Gly Gly Gly Thr Lys Leu Glu Ile Lys

1 5 101 5 10

Claims

1. A monoclonal antibody of anti GPC3, can discern GPC3 protein specifically, the antibody is mouse antibody, produced by mouse hybridoma cell line GPC3-T with the collection number of CCTCC NO: C2020109, CDR1 of the heavy chain variable region is as the amino acid sequence shown in SEQ ID NO:46, CDR2 is as the amino acid sequence shown in SEQ ID NO:47, CDR3 is as the amino acid sequence shown in SEQ ID NO: 48; the light chain variable region CDR1 is shown as the amino acid sequence of SEQ ID NO:53, CDR2 is shown as the amino acid sequence of SEQ ID NO:54, and CDR3 is shown as the amino acid sequence of SEQ ID NO: 55;

wherein, the mouse hybridoma cell strain with the preservation number of CCTCC NO: C2020109 is generated by GPC3 protein expressed by the gene with the nucleotide sequence of SEQ ID NO:1 in eukaryote.

2. The use of the monoclonal antibody against GPC3 according to claim 1 for the preparation of a pharmaceutical preparation for the diagnosis or treatment of primary liver cancer.