CN116903745B - 一种抗人Claudin18.2的单克隆抗体及其应用 - Google Patents
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Abstract
本发明公开了一种抗人Claudin18.2的单克隆抗体及其应用。该抗体具有如SEQ ID NO:2~4所示的轻链互补决定区,以及如SEQ ID NO:5~7所示的重链互补决定区。本发明提供的单克隆抗体能与人Claudin18.2蛋白特异结合,且与人Claudin18.1蛋白无交叉反应,效价高,亲和力好,能够用于特异检测人Claudin18.2蛋白,通过免疫组化方法检测待检测标本组织中的Claudin18.2的表达情况,检测结果可以作为临床医生的诊断依据之一,为临床肿瘤的诊断起预后判断的作用,帮助临床肿瘤的诊断、分类、预后判断以及疗效评估。
Description
技术领域
本发明属于生物技术领域,具体涉及一种分泌抗人Claudin18.2单克隆抗体的杂交瘤细胞及应用。
背景技术
Claudin蛋白是在鸡的肝脏中发现的一类存在于细胞上的4次跨膜蛋白,是构成紧密连接的重要组成部分,其具有黏附细胞、维持细胞极性、调节细胞通透性以及参与细胞增殖和分化的调节等功能。Claudin18是目前Claudin 蛋白家族中研究最为深入的一种,包括Claudin18.1和 Claudin18.2两种亚型。正常情况下Claudin18.2蛋白主要表达在胃黏膜上皮细胞,而不表达于人体其他健康组织中。但其在胃癌、食管癌、胰腺癌等癌变组织中表达明显上调,并能够参与肿瘤细胞的增殖、分化及转移。目前,利用免疫组织化学方法对关键肿瘤相关蛋白进行组织学检测是关系到肿瘤精准诊疗的重要节。如何准确、快速的检测出各肿瘤组织Claudin18.2的表达情况是靶向免疫治疗的关键。
由于Claudin18.1和Claudin18.2氨基酸序列仅有极小的差异,目前市面上大多数claudin18.2抗体并不能特异性识别Claudin18.2而不识别Claudin18.1,因此,一种抗人Claudin18.2的单克隆抗体及其应用亟待提出。
发明内容
为解决现有技术存在的缺陷,本发明提供一种分泌抗Claudin18.2单克隆抗体的杂交瘤细胞及应用,该单克隆抗体不与任何其他Claudin家族成员结合,其中包括Claudin18.1蛋白亚型,并且目前尚没有任何其它Claudin18.2抗体经过大量组织标本的免疫组化验证并显示出可以通过对Claudin18.2的组织细胞表达水平进行检测来达到对胃癌恶性度及患者生存情况进行预后预判的目的。
为了解决上述技术问题,本发明提供了如下的技术方案:
本发明的第一目的提供一种抗人Claudin18.2的单克隆抗体,所述单克隆抗体能够特异性识别人Claudin18.2蛋白,所述识别位点的氨基酸序列如SEQ ID NO:1所示,所述单克隆抗体具有如SEQ ID NO:2所示的轻链互补决定区CDR1、如SEQ ID NO:3所示的轻链互补决定区CDR2和如SEQ ID NO:4所示的轻链互补决定区CDR3、以及如SEQ ID NO:5所示的重链互补决定区CDR1、如SEQ ID NO:6所示的重链互补决定区CDR2和如SEQ ID NO:7所示的重链互补决定区CDR3。
优选的,所述单克隆抗体包括重链可变区和轻链可变区,所述轻链可变区的氨基酸序列如SEQ ID NO:8所示,所述重链可变区的氨基酸序列如SEQ ID NO:9所示。
本发明的第二目的提供一种核酸组合物,包括编码抗人Claudin18.2的单克隆抗体的核酸分子。
本发明的第三目的提供一种检测试剂盒,所述试剂盒中还含有柠檬酸盐缓冲液、内源性过氧化酶阻断剂、酶标羊抗鼠IgG聚合物、DAB缓冲液、DAB底物、DAB显色剂、PBS磷酸盐缓冲液、抗人Claudin18.2的单克隆抗体。
本发明的第四目的提供一种抗人Claudin18.2的单克隆抗体在制备用于免疫组化检测的试剂中的应用。
本发明相较于现有技术,具有以下有益效果:
本发明提供的单克隆抗体能与Claudin18.2蛋白特异结合,不与Claudin18.1产生交叉反应,效价高,亲和力好,能够用于特异检测人Claudin18.2蛋白,并用于胃癌的免疫组化检测。本发明提供抗体为核心的免疫组化检测方法,能很好地检测胃癌组织细胞中Claudin18.2的表达量和表达位置,便于从免疫组化图中直接判读Claudin18.2在癌组织细胞中的定位和表达情况,并以此为依据判别胃癌的个体化异质性及其生存预后。
附图说明
图1是本发明实施例1中SDS-PAGE结果示意图;
图2是本发明实施例1中蛋白质印迹分析结果示意图;
图3是本发明实施例2中SDS-PAGE结果示意图;
图4是本发明实施例2中蛋白质印迹分析结果示意图;
图5是本发明实施例5中Claudin18.2在浆中呈弱阳性着色的示意图;
图6是本发明实施例5中Claudin18.2在浆中呈强阳性着色的示意图;
图7 是本实施例7中免疫印迹法确认抗体特异性的结果示意图。
具体实施方式
以下结合附图对本发明的优选实施例进行说明,应当理解,此处所描述的优选实施例仅用于说明和解释本发明,并不用于限定本发明。
本发明中基因Claudin18.2的GeneBank国际通用序列编号为:NC_000003.12,基因Claudin18.2编码蛋白的GeneBank国际通用序列编号为:NP_001002026.1。
实施例1:Claudin18.2的重组蛋白表达、纯化及鉴定
从胃癌组织中利用RT-PCR的方法,利用Claudin18.2-F、Claudin18.2-R为引物克隆Claudin18.2基因(其中,Claudin18.2-F的基因序列如SEQ ID NO:10所示,Claudin18.2-R的基因序列如SEQ ID NO:11所示,Claudin18.2基因的基因序列如SEQ ID NO:12所示),然后将克隆的Claudin18.2基因插入含有His标签的改造过pc-DNA3.1载体上,得Claudin18.2-pc DNA3.1。然后将Claudin18.2-pc DNA3.1转化DH5α,挑取阳性克隆,将挑取的阳性克隆扩大培养后,提取重组质粒Claudin18.2-pc DNA3.1,然后以HindⅢ、BamHI进行酶切分析,结果显示得到了与预期大小相符的酶切片段。将酶切正确的重组Claudin18.2-pc DNA3.1经测序确认基因正确后利用Lipofectamine2000转染入292T细胞中,经过G418筛选阳性克隆,以含10%胎牛血清的DMEM培养基培养(37℃,5%CO2),收集培养上清并纯化,细胞表达产物经SDS-PAGE分析,结果如图1所示。结果显示,发现在26KD左右有特异表达带。用anti-6x-His 抗体进行蛋白质印迹分析,结果如图2所示,仅有一条特异性条带,表明蛋白为目的蛋白。
利用镍柱纯化细胞培养上清,具体方法为收集细胞培养液,1200 rpm离心分离细胞,收集上清,并利用0.45μm 滤器过滤上清液。将滤液上样到5ml Ni-NTA 柱上,经5倍柱体积10mM咪唑溶液清洗,然后用300mM咪唑洗脱,收集各管洗脱蛋白溶液。SDS-PAGE鉴定蛋白纯度并定量,获得的蛋白纯度达到90%,浓度为1.25mg/ml。
实施例2:Claudin18.2单克隆抗体的制备、纯化及鉴定
用实施例1纯化的Claudin18.2重组蛋白利用皮下注射方法免疫BALB/c小鼠,每只小鼠每次免疫50μg抗原,免疫体积为100μl。首次免疫Claudin18.2与弗氏完全佐剂1:1混合每隔两周免疫一次,Claudin18.2与弗氏不完全完全佐剂1:1混合,共免疫3次;最后经过Claudin18.2(不含佐剂)腹腔加强免疫(30μg),3天后取免疫小鼠的脾脏细胞,然后利用PEG-4000融合免疫小鼠脾脏细胞和小鼠的骨髓瘤细胞系SP2/0,利用HAT选择培养基在96孔细胞培养板上选择杂交瘤细胞,并利用ELISA方法鉴定产生抗Claudin18.2蛋白且与Claudin18.1 不产生交叉反应的杂交瘤细胞株4G7,然后利用转瓶培养获得的杂交瘤细胞株,收集细胞培养上清,利用0.45um的滤器过滤溶液,加入终浓度为50%的硫酸铵固体,4℃搅拌均匀后,静置1小时,12000 rpm收集沉淀,并用50mM PBS溶液重溶沉淀。将得到粗纯化抗体在AKTA 纯化系统上,以1ml/min 流速上样到Protein A-Sepharose亲和层析柱中,用5个柱体积的结合缓冲液(50mM PBS pH7.0)清洗,然后用0.1M 甘氨酸-盐酸溶液 pH2.7洗脱抗体(每个收集管预先加入1M pH 9.0 Tris缓冲液中和),得到目的抗体。然后SDS-PAGE及Western Blotting鉴定分离纯化的单抗,并命名为4G7单克隆抗体。SDS-PAGE结果如图3所示,纯化后的单克隆抗体仅有50kd处的重链和25kd处的轻链,灰度分析显示,抗体纯度达到95%。将重组蛋白Claudin18.2 以5μg每孔上样,上述单克隆抗体1:10000 稀释, 蛋白质印迹分析结果如图4所示,显示抗体特异性与重组蛋白反应。
实施例3:Claudin18.2免疫组化抗体筛选
取胃癌及癌旁组织切片脱蜡至水,抗原修复,内源过氧化物酶阻断,以1:200分别滴加上述制备得到的鼠抗人Claudin18.2单克隆抗体作为一抗,4℃孵育过夜。缓冲液洗三次,每次5min。滴加酶标羊抗鼠IgG二抗 ,在室温下孵育30min。缓冲液洗三次,每次5min。DAB显色,复染,脱水封片后显微镜下观察染色情况。结果显示4G7单克隆抗体特异性较高,与酶标抗体无特异性交叉反应。
实施例4:Claudin18.2免疫组化试剂盒
试剂盒组分:试剂1:柠檬酸盐缓冲液;试剂2:内源性过氧化酶阻断剂;试剂3:鼠抗人Claudin18.2单克隆抗体;试剂4:酶标羊抗鼠IgG二抗;试剂5A:DAB缓冲液;试剂5B:DAB底物;试剂5C:DAB显色剂;其他组成:PBS磷酸盐缓冲液。
实施例5:Claudin18.2免疫组化试剂盒检测样本
胃癌切片取自上海交通大学附属仁济医院肿瘤组织标本库,组织切片脱蜡至水,抗原修复,内源过氧化物酶阻断,以1:200滴加鼠抗人Claudin18.2单克隆抗体作为一抗,4℃孵育过夜。缓冲液洗三次,每次5min。滴加酶标羊抗鼠IgG二抗 ,在室温下孵育30min。缓冲液洗三次,每次5min。DAB显色,复染,脱水封片后显微镜下观察染色情况。
结果:胃癌患者组织细胞中Claudin18.2在浆中呈不同程度的着色,浆中弱阳性着色见图5,浆中强阳性着色见图6。采用本发明提供抗体为核心的免疫组化检测方法,能很好地检测胃癌组织细胞中Claudin18.2的表达量和表达位置,便于从免疫组化图中直接判读Claudin18.2在癌组织细胞中的定位和表达情况,并以此为依据为胃癌患者的个体化治疗方案提供指导。
实施例6:ELISA法确认抗体特异性
分别以CBS缓冲液100μl体积包被5μg/ml的Claudin18.1、Claudin18.2和Claudin16蛋白至微孔板4℃过夜,封闭后将4株抗体稀释至1ug/ml后加入到微孔反应板,然后加入羊抗鼠 IgG-HRP(0.5ug/ml),结果如表1所示,4G7特异性识别Claudin18.2,且不与Claudin18.1及Claudin16发生交叉反应。
表1 抗体与Claudin18.1交叉反应性验证
将Claudin18.1、Claudin18.2蛋白每孔5ug上样,进行SDS-PAGE电泳,并转印至硝酸纤维素膜(NC)上,封闭后将4G7抗体稀释至0.1ug/ml室温孵育2h,然后加入羊抗鼠 IgG-HRP(1:10000稀释)室温孵育2h,加入增强化学发光 (ECL)底物进行曝光反应,结果如图7所示,抗体4G7特异性识别Claudin18.2,不与Claudin18.1交叉反应。
最后应说明:以上所述仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (5)
1.一种抗人Claudin18.2的单克隆抗体,其特征在于,所述单克隆抗体能够特异性识别人Claudin 18.2蛋白,识别位点的氨基酸序列如SEQ ID NO:1所示,所述单克隆抗体具有如SEQ ID NO:2所示的轻链互补决定区CDR1、如SEQ ID NO:3所示的轻链互补决定区CDR2和如SEQ ID NO:4所示的轻链互补决定区CDR3、以及如SEQ ID NO:5所示的重链互补决定区CDR1、如SEQ ID NO:6所示的重链互补决定区CDR2和如SEQ ID NO:7所示的重链互补决定区CDR3。
2.根据权利要求1所述的抗人Claudin18.2的单克隆抗体,其特征在于,所述单克隆抗体包括重链可变区和轻链可变区,所述轻链可变区的氨基酸序列如SEQ ID NO:8所示,所述重链可变区的氨基酸序列如SEQ ID NO:9所示。
3.一种核酸组合物,其特征在于,包括编码权利要求1或2所述抗人Claudin18.2的单克隆抗体的核酸分子。
4.一种检测试剂盒,其特征在于,所述试剂盒中还含有柠檬酸盐缓冲液、内源性过氧化酶阻断剂、酶标羊抗鼠IgG聚合物、DAB缓冲液、DAB底物、DAB显色剂、PBS磷酸盐缓冲液、如权利要求1或2所述的抗人Claudin18.2的单克隆抗体。
5.一种如权利要求1或2所述的抗人Claudin18.2的单克隆抗体在制备用于免疫组化检测的试剂中的应用。
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU2012202225A1 (en) * | 2005-11-24 | 2012-05-10 | Ganymed Pharmaceuticals Ag | Monoclonal antibodies against claudin-18 for treatment of cancer |
AU2014208256A1 (en) * | 2005-11-24 | 2014-09-18 | Astellas Pharma Inc. | Monoclonal antibodies against claudin-18 for treatment of cancer |
US10421817B1 (en) * | 2019-01-17 | 2019-09-24 | Beijing Mabworks Biotech Co., Ltd. | Antibodies binding human Claudin 18.2 and uses thereof |
WO2021115303A1 (zh) * | 2019-12-11 | 2021-06-17 | 上海复宏汉霖生物技术股份有限公司 | 一种抗Claudin18.2单克隆抗体、其制备方法及用途 |
CN114751984A (zh) * | 2021-09-03 | 2022-07-15 | 深圳市先康达生命科学有限公司 | 靶向人Claudin18.2蛋白的单克隆抗体及其应用 |
Family Cites Families (2)
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CN110857322A (zh) * | 2018-08-22 | 2020-03-03 | 瑞阳(苏州)生物科技有限公司 | 抗人claudin 18.2单克隆抗体及其应用 |
MX2022002111A (es) * | 2019-08-20 | 2022-03-17 | Suzhou Transcenta Therapeutics Co Ltd | Novedosos anticuerpos anti-cldn18.2. |
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Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU2012202225A1 (en) * | 2005-11-24 | 2012-05-10 | Ganymed Pharmaceuticals Ag | Monoclonal antibodies against claudin-18 for treatment of cancer |
AU2014208256A1 (en) * | 2005-11-24 | 2014-09-18 | Astellas Pharma Inc. | Monoclonal antibodies against claudin-18 for treatment of cancer |
US10421817B1 (en) * | 2019-01-17 | 2019-09-24 | Beijing Mabworks Biotech Co., Ltd. | Antibodies binding human Claudin 18.2 and uses thereof |
WO2021115303A1 (zh) * | 2019-12-11 | 2021-06-17 | 上海复宏汉霖生物技术股份有限公司 | 一种抗Claudin18.2单克隆抗体、其制备方法及用途 |
CN114751984A (zh) * | 2021-09-03 | 2022-07-15 | 深圳市先康达生命科学有限公司 | 靶向人Claudin18.2蛋白的单克隆抗体及其应用 |
Non-Patent Citations (2)
Title |
---|
Claudin蛋白在胃癌中的研究进展;车娟娟;王婧;曹邦伟;;医学综述(17);全文 * |
CLDN18.2蛋白在恶性肿瘤治疗中的研究进展;徐良额;何天阳;张丽;陆一丹;罗聪;;中国肿瘤临床(06);全文 * |
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