CN116925231B - 一种抗mmp19蛋白的抗体及其应用 - Google Patents
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Abstract
本发明公开了一种抗MMP19蛋白的抗体及其应用,所述抗体的重链可变区的氨基酸序列如SEQ ID NO:4所示,轻链可变区的氨基酸序列如SEQ ID NO:1所示,本发明提供的抗体对MMP19具有良好的亲和力和特异性,能够用于各种受试者来源的待测样本中MMP19的有效快速检测中,具有良好的应用前景。
Description
技术领域
本发明属于抗体药物技术领域,具体地,本发明涉及一种抗MMP19蛋白的抗体及其应用。
背景技术
基质金属蛋白酶(Matrix metalloproteinase,MMPs)是体内普遍存在的锌离子依赖性蛋白酶,参与人体多种生理和病理过程,如降解细胞外基质、抗炎症反应、抗血管生成及恶性肿瘤的进展和转移等。MMPs包括大约24种不同类型的MMP,分为六个亚家族:胶原酶(MMP-1、MMP-8和MMP-13)、明胶酶(MMP-2和MMP-9)、间质溶解素(MMP-3、MMP-10和MMP-11)、基质溶解素(MMP-7和MMP-26)、膜型金属蛋白酶(MT1-6-MMPs,MMP-14、MMP-15、MMP-16、MMP-17、MMP-24和MMP-25)和其他MMP(MMP-12、MMP-18、MMP-19、MMP-20、MMP-21、MMP-22、MMP-23、MMP-27和MMP-28)。除MMP-7和MMP-26外,所有MMPs都具有与催化结构域连接的血脂样结构域,介导MMPs与底物、内源性抑制剂和细胞表面分子的相互作用。
基质金属蛋白酶-19(MMP19)是MMPs家族的重要成员之一,也被称为基质金属蛋白酶RASI,是一种由MMP19基因编码的酶。最先从自身免疫性类风湿关节炎患者中分离得到,随后在机体多种组织中被陆续发现,其表达的程度不一。MMP19不仅具有大多数MMPs家族中降解细胞外基质(extracellular matrix,ECM)的作用,而且在肿瘤血管生成、癌细胞生长转移过程中起负调节作用,其既能明显抑制人微血管内皮细胞增殖而减少毛细血管样结构生成,又能分离与ECM结合的血管内皮生长因子亚群,以减弱ECM参与新生血管的作用,这与多数MMPs介导的功能不同。此外,MMP19参与结直肠癌、肺癌、胃癌、胰腺癌等多种癌症的进展和转移。近期研究显示,MMP19还可作为肾脏早期损伤相关和焦虑抑郁症筛选药物和诊断的新标志物。目前,关于靶向MMP19的抗体的研究相对较少,因此,本领域迫切需要一种对MMP19具有较好亲和力和特异性、能够有效快速检测MMP19的单克隆抗体产品。
发明内容
有鉴于此,本发明的目的在于提供一种抗MMP19蛋白的抗体及其应用。
为了实现上述目的,本发明采用了如下技术方案:
本发明的第一方面提供了一种抗MMP19蛋白的抗体。
进一步,所述抗体包含重链可变区和轻链可变区;
所述重链可变区中HCDR1、HCDR2、HCDR3的氨基酸序列为如SEQ ID NO:4所示的重链可变区中的CDR1、CDR2、CDR3;
所述轻链可变区中LCDR1、LCDR2、LCDR3的氨基酸序列为如SEQ ID NO:1所示的轻链可变区中的CDR1、CDR2、CDR3。
进一步,所述HCDR1、HCDR2、HCDR3的氨基酸序列分别如SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7所示;
所述LCDR1、LCDR3的氨基酸序列分别如SEQ ID NO:2、SEQ ID NO:3所示;
所述LCDR2的氨基酸序列为LVS。
在一些实施方案中,与本发明所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2、LCDR3对应的氨基酸序列具有至少90%(包括90%、91%、92%、93%、94%、95%、96%、97%、98%、99%)序列同源性的氨基酸序列也均在本发明的保护范围内,所述具有至少90%序列同源性的氨基酸序列包括通过对亲本序列进行一个或者多个氨基酸或核苷酸缺失、插入或替换突变获得。
在一些实施方案中,采用任何CDR编号方案(现有的CDR编号方案或将来产生的新的CDR编号方案)分别对如SEQ ID NO:4所示的重链可变区中的CDR1、CDR2、CDR3进行定义、对如SEQ ID NO:1所示的轻链可变区中的CDR1、CDR2、CDR3进行定义得到的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2、LCDR3对应的氨基酸序列或核苷酸序列均在本发明的保护范围内,其对应的抗体同样包含在本发明的保护范围内。
在一些实施方案中,所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2、LCDR3是根据IMGT编号方案、Chothia编号方案、Kabat编号方案、Martin(增强型Chothia)编号方案、AbM编号方案、Aho编号方案中的任意一种定义或任意多种(两种或两种以上)组合定义得到的,经上述定义方式定义得到的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2、LCDR3对应的抗体同样包含在本发明的保护范围内。
本领域普通技术人员可以很容易地采用已知的方法,例如采用定向点突变的方法,对本发明提供的抗体对应的核苷酸序列进行突变。那些经过人工修饰的、具有与本发明所述的抗体对应的核苷酸序列75%或75%以上同源性的核苷酸,只要编码本发明第一方面所述的抗体,均是衍生于本发明的核苷酸序列并且等同于本发明的序列,同样包含在本发明的保护范围内。
本发明的第二方面提供了一种核酸分子。
进一步,所述核酸分子编码本发明第一方面所述的抗体。
在一些实施方案中,所述核酸分子是分离或纯化的。DNA分子的序列可以用常规技术,或利用杂交瘤技术获得。一旦获得了有关的序列,就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体,再转入细胞,然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。此外,还可用人工合成的方法来合成有关序列,尤其是片段长度较短时。通常,通过先合成多个小片段,然后再进行连接可获得序列相对较长的片段。
本发明的第三方面提供了一种表达载体。
进一步,所述表达载体包含本发明第二方面所述的核酸分子。
在一些实施方案中,所述表达载体中的核酸分子与启动子可操作性地连接,所述启动子的实例包括但不限于:tac启动子、lac启动子、lacUV5启动子、lpp启动子、pLλ启动子、pRλ启动子、rac5启动子、amp启动子、recA启动子、SP6启动子、trp启动子、T7启动子、SV40启动子、CMV启动子、MMTV启动子。
在一些实施方案中,所述载体的实例包括但不限于:质粒载体、粘粒载体、病毒载体,其中,所述病毒载体包括但不限于:噬菌体载体、腺病毒载体、逆转录病毒载体、腺相关病毒载体、慢病毒载体。
在一些实施方案中,本发明对载体并没有特别的限制,其选择取决于所期望的功能。载体的非限制性实例包括质粒载体、病毒来源的载体(例如慢病毒载体、逆转录病毒载体、腺病毒载体、腺相关病毒载体、痘病毒载体、疱疹病毒载体、杆状病毒载体、乳头状瘤病毒载体、乳头多瘤空泡病毒载体)、噬菌体载体和其他常规用于例如遗传工程的载体。可基于本领域技术人员所熟知的方法构建各种质粒和载体。
在一些实施方案中,所述表达载体还包含适当启动子或者控制序列的载体。这些表达载体可以用于转化适当的宿主细胞,以使其能够表达蛋白质。宿主细胞可以是原核细胞,如细菌细胞;或是低等真核细胞,如酵母细胞;或是高等真核细胞,如哺乳动物细胞。代表性的实例有:细菌细胞如大肠杆菌,链霉菌属;鼠伤寒沙门氏菌;真菌细胞如酵母;植物细胞;昆虫细胞如果蝇S2或Sf9;动物细胞如CHO、COS7、NSO或Bowes黑素瘤细胞等。特别适用于本发明的宿主细胞是真核宿主细胞,尤其是哺乳动物细胞,例如CHO细胞、293细胞等。
本发明的第四方面提供了一种宿主细胞。
进一步,所述宿主细胞包含本发明第三面所述的表达载体。
进一步,所述宿主细胞为保藏号为CGMCC NO. 45660的杂交瘤细胞。
在一些实施方案中,所述真核细胞或原核细胞能够用作表达本发明第一方面所述抗体的宿主细胞,此类宿主细胞是本领域技术人员所熟知的,并且许多类型的宿主细胞可从美国典型培养物保藏中心(American Type Culture Collection,ATCC)获得。所述宿主细胞优选为哺乳动物宿主细胞,所述哺乳动物宿主细胞包括骨髓瘤细胞,例如SP2/0细胞和NS0细胞以及中国仓鼠卵巢(CHO)细胞、杂交瘤细胞系及其它可用于表达本发明第一方面所述抗体的哺乳动物宿主细胞。另外,用于表达抗体的特别有用的宿主细胞是人细胞系PER.C6,其公开于WO0063403A2,其能够产生比常规哺乳动物细胞系(例如CHO、COS、Vero、Hela、BHK和SP2-细胞系)高2-200倍的重组蛋白。
本发明的第五方面提供了如下任一种物质:
(1) 一种抗体衍生物,所述抗体衍生物包含本发明第一方面所述的抗体直接或间接偶联至可检测标记物上形成的复合物;
(2) 一种检测试剂,所述检测试剂包含本发明第一方面所述的抗体、本发明第二方面所述的核酸分子、本发明第三方面所述的表达载体、本发明第四方面所述的宿主细胞和/或所述抗体衍生物;
(3) 一种检测试剂盒,所述检测试剂盒包含本发明第一方面所述的抗体、本发明第二方面所述的核酸分子、本发明第三方面所述的表达载体、本发明第四方面所述的宿主细胞、所述抗体衍生物和/或所述检测试剂;
(4) 一种检测试纸条,所述检测试纸条包含本发明第一方面所述的抗体、本发明第二方面所述的核酸分子、本发明第三方面所述的表达载体、本发明第四方面所述的宿主细胞、所述抗体衍生物和/或所述检测试剂;
(5) 一种药物组合物,所述药物组合物包含本发明第一方面所述的抗体、本发明第二方面所述的核酸分子、本发明第三方面所述的表达载体和/或本发明第四方面所述的宿主细胞。
在一些实施方案中,所述抗体衍生物中包含的可检测标记物包括但不限于:生物发光剂、顺磁性离子、酶、光敏诊断剂、放射性核素、化学发光剂。
在一些实施方案中,所述生物发光剂包括但不限于:荧光素、荧光素酶、水母发光蛋白;所述顺磁性离子包括但不限于:铬(III)、镍(II)、铜(II)、钕(III)、钐(III)、镱(III)、锰(II)、铁(III)、铁(II)、钴(II)、钆(III)、钒(II);所述酶包括但不限于:辣根过氧化物酶、过氧化氢酶、碱性磷酸酯酶、脲酶、葡萄糖氧化酶、β-D-半乳糖苷酶、或葡萄糖淀粉酶;所述光敏诊断剂包括但不限于:二羟基硅酞菁、原卟啉、血卟啉、亚甲基蓝、光卟啉;所述放射性核素包括但不限于:68Ga、86Y、90Y、89Zr、110In、111In、177Lu、18F、52Fe、62Cu、64Cu、31I、154-158Gd、32F、11C、67Cu、67Ga、94mTc、94Tc、99mTc、120I、123I、124I、125I、113N、15O、186Re、188Re;所述化学发光剂包括但不限于:芳族吖啶酯、咪唑、鲁米诺、异鲁米诺、吖啶盐、草酸酯。
在一些实施方案中,所述药物组合物还包含药学上可接受的赋形剂,所述药学上可接受的赋形剂在Remington's Pharmaceutical Sciences(19th ed.,1995)中有详细的记载,这些物质根据需要用于帮助配方的稳定性或有助于提高活性或活性物质的生物有效性或在口服的情况下产生可接受的口感或气味,在这种药物组合物中可以使用的制剂可以是其原始化合物本身的形式,或任选地使用其药物学可接受的盐的形式。如此配制得到的药物组合物根据需要可选择本领域技术人员已知的任何适当的方式将药物组合物进行给药。
在一些实施方案中,所述药物组合物可具有选自如下的任一种制剂:片剂、丸剂、粉剂、颗粒剂、胶囊、悬浮液、溶液剂、乳剂、糖浆、灭菌水溶液剂、非水溶液剂、悬浮剂、乳剂、冻干制剂和栓剂。此外,可以一次或多次施用,此时,以液体制剂、粉剂、气雾剂、胶囊、阴道片剂、胶囊或栓剂的形式施用本发明所述的药物组合物。
在一些实施方案中,所述药物组合物的施用方式包括但不限于:腹膜内、静脉内、肌内、皮下、皮内、经口、局部、鼻内、肺内、直肠内等。当口服施用时,可配制成保护药物组合物中的活性成分以防止其在胃中降解的包衣。此外,可通过任何能够转移至靶组织的装置施用活性成分。
在具体实施方案中,本发明提供的药物组合物可根据实际需要制成各种剂型,并可由临床医师根据受试者的种类、年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的剂量进行施用。给药方式例如可以采用注射或其它本领域技术人员公知的任何合适的给药方式。
本发明的第六方面提供了如下任一种方法,所述方法包括:
(1) 一种生产本发明第一方面所述的抗体的方法,所述方法包括如下步骤:在适合的条件下,培养本发明第四方面所述的宿主细胞,从宿主细胞培养产物中分离纯化得到本发明第一方面所述的抗体;
(2) 一种制备本发明第四方面所述的宿主细胞的方法,所述方法包括如下步骤:将本发明第二方面所述的核酸分子或本发明第三方面所述的表达载体导入到宿主细胞中,得到本发明第四方面所述的宿主细胞;
(3) 一种体外非治疗目的地抑制MMP19蛋白的方法,所述方法包括如下步骤:将本发明第一方面所述的抗体、本发明第二方面所述的核酸分子和/或本发明第三方面所述的表达载体导入到生物体细胞中,通过表达本发明第一方面所述的抗体抑制MMP19蛋白的活性;
(4) 一种非诊断和非治疗目的地检测MMP19蛋白的方法,所述方法包括如下步骤:将待测样品与本发明第一方面所述的抗体、本发明第五方面中所述的抗体衍生物和/或本发明第五方面中所述的检测试剂接触,检测MMP19蛋白与抗体免疫复合物的形成。
在一些实施方案中,将所述核酸分子导入到宿主细胞中的方法是本领域技术人员所熟知的,包括但不限于:物理方法、化学方法、生物方法。
在具体实施方案中,所述物理方法包括磷酸钙沉淀、脂质转染法、粒子轰击、微注射、电穿孔等等。所述化学方法包括胶体分散系统,诸如大分子复合物、纳米胶囊、微球、珠;和基于脂质的系统,包括水包油乳剂、胶束、混合胶束和脂质体。用作体外和体内传递工具(delivery vehicle)的示例性胶体系统为脂质体(例如,人造膜囊)。所述生物方法包括使用DNA和RNA载体。病毒载体,特别是逆转录病毒载体,已经成为最广泛使用的将基因插入哺乳动物例如人细胞的方法。其他病毒载体可源自慢病毒、痘病毒、单纯疱疹病毒I、腺病毒和腺伴随病毒等等。
在一些实施方案中,所述待测样品可选自待测受试者来源的尿液、血液、血清、血浆、唾液、腹水、非组织缔合的细胞、组织、组织学制备物等,本发明对待测样品类型并无特别限制。
在一些实施方案中,所述受试者包括人类和非人类动物。非人动物包括所有脊椎动物(例如哺乳动物和非哺乳动物)例如非人灵长类(例如食蟹猴)、绵羊、狗、牛、鸡、两栖动物和爬行动物。在具体的实施方案中,所述受试者优选为人。
本发明的第十方面提供了如下任一种应用,所述应用包括:
(1) 本发明第四方面所述的宿主细胞在制备本发明第一方面所述的抗体中的应用;
(2) 本发明第一方面所述的抗体、本发明第二方面所述的核酸分子、本发明第三方面所述的表达载体和/或本发明第四方面所述的宿主细胞在制备用于检测MMP19蛋白的抗体衍生物中的应用;
(3) 本发明第一方面所述的抗体、本发明第二方面所述的核酸分子、本发明第三方面所述的表达载体、本发明第四方面所述的宿主细胞和/或本发明第五方面中所述的抗体衍生物在制备用于检测MMP19蛋白的检测试剂中的应用;
(4) 本发明第一方面所述的抗体、本发明第二方面所述的核酸分子、本发明第三方面所述的表达载体、本发明第四方面所述的宿主细胞、本发明第五方面中所述的抗体衍生物和/或本发明第五方面中所述的检测试剂在制备用于检测MMP19蛋白的检测试剂盒中的应用;
(5) 本发明第一方面所述的抗体、本发明第二方面所述的核酸分子、本发明第三方面所述的表达载体、本发明第四方面所述的宿主细胞、本发明第五方面中所述的抗体衍生物和/或本发明第五方面中所述的检测试剂在制备用于检测MMP19蛋白的检测试纸条中的应用;
(6) 本发明第一方面所述的抗体、本发明第二方面所述的核酸分子、本发明第三方面所述的表达载体、本发明第四方面所述的宿主细胞、本发明第五方面中所述的抗体衍生物、本发明第五方面中所述的检测试剂、本发明第五方面中所述的检测试剂盒和/或本发明第五方面中所述的检测试纸条在制备用于诊断和/或辅助诊断MMP19蛋白相关疾病的产品中的应用;
(7) 本发明第一方面所述的抗体、本发明第二方面所述的核酸分子、本发明第三方面所述的表达载体和/或本发明第四方面所述的宿主细胞在制备用于治疗和/或预防MMP19蛋白相关疾病的药物中的应用。
进一步,所述MMP19蛋白相关疾病包括结直肠癌、肺癌、胃癌、胰腺癌、甲状腺乳头状癌、卵巢癌、肝癌、骨肉瘤、胃癌、神经胶质瘤、类风湿关节炎、系统性红斑狼疮、多发性硬化症。
在一些实施方案中,只要与MMP19蛋白表达相关的疾病均在本发明的保护范围内,并不局限于本发明所列举出的具体上述疾病类型。
此外,本发明还提供了一种诊断和/或辅助诊断MMP19蛋白相关疾病的方法,所述方法包括如下步骤:采用本发明第一方面所述的抗体、本发明第五方面中所述的抗体衍生物、本发明第五方面中所述的检测试剂、本发明第五方面中所述的检测试剂盒和/或本发明第五方面中所述的检测试纸条对受试者来源的待测样品进行检测,通过抗原抗体反应来检测待测样品中MMP19蛋白的存在与否,以诊断和/或辅助诊断受试者是否患有MMP19蛋白相关疾病和/或患MMP19蛋白相关疾病的风险。
相对于现有技术,本发明具有的优点和有益效果如下:
本发明首次公开了一种用于检测MMP19蛋白的单克隆抗体,所述抗体的重链可变区中HCDR1、HCDR2、HCDR3的氨基酸序列为如SEQ ID NO:4所示的重链可变区中的CDR1、CDR2、CDR3,所述抗体的轻链可变区中LCDR1、LCDR2、LCDR3的氨基酸序列为如SEQ ID NO:1所示的轻链可变区中的CDR1、CDR2、CDR3。本发明提供的抗体对MMP19具有良好的亲和力和特异性,能够用于各种受试者来源的待测样本中MMP19的有效快速检测中,具有良好的应用前景。
附图说明
图1为抗MMP19单克隆抗体SDS-PAGE电泳分析结果图,图中标号M为Marker,标号1为纯化后的4C9B12D7D12细胞株。
图2为抗MMP19单克隆抗体亚类鉴定结果图,4C9B12D7D12细胞株为IgG1型。
图3为抗MMP19单克隆抗体Western Blot免疫检测真核表达MMP19蛋白的结果图,图中标号1为转染pcDNA3.4空载体的293细胞裂解液,标号2为转染pcDNA3.4-MMP19质粒的293细胞裂解液。
具体实施方式
生物材料保藏说明:本发明的单克隆抗体杂交瘤细胞株:小鼠杂交瘤细胞株4C9B12D7D12,保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏中心登记入册编号为CGMCC No. 45660,保藏日期为:2023年08月17日。中国微生物菌种保藏管理委员会普通微生物中心,地址为:北京市朝阳区北辰西路1号院3号,邮编100101。
下面结合具体实施例,进一步阐述本发明,具体实施例仅用于解释本发明,而不能理解为对本发明的限制。本领域的普通技术人员可以理解为:在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由权利要求及其等同物限定。下列实施例中未注明具体条件的实验方法,通常按照常规条件或按照厂商所建议的条件实施检测。
实施例1 免疫原制备
根据Genebank中公布的人MMP19蛋白全长基因序列,由北京擎科生物科技有限公司合成全长基因,并连接入pET-30a质粒中,获得重组质粒pET-30a-MMP19。将重组质粒转化入Rosetta感受态细胞,挑取单菌落于2 mL含卡那霉素的LB液体培养基中,37℃振荡培养过夜,次日接种于200 mL新鲜的LB液体培养基中,37℃、180 rpm培养4 h至对数生长期,加200μL 1 mol/L的IPTG诱导液,37℃诱导12-14 h。4℃,6000 rpm离心10 min收集诱导后的菌体;用25 mmol/L Tris-HCl(pH=8.5)重悬菌体,冰浴超声;进行SDS-PAGE凝胶电泳,分析MMP19蛋白主要以包涵体形式表达。将pET-30a-MMP19菌液扩大培养,用Ni柱进行亲和层析纯化蛋白,用含25 mM咪唑、250 mM咪唑的6M脲1 ‰ β-巯基乙醇25 mM Tris-HCl(pH=8.5)溶液洗脱,分别收集蛋白洗脱峰,电泳分析纯化产物(电泳结果图如图1所示),取250 mM咪唑洗脱蛋白透析后作为免疫原(MMP19抗原)。
实施例2 杂交瘤细胞株建立
取纯化后MMP19蛋白作为免疫原,采用8周龄BALB/c雌性小鼠,抗原加MnJ胶体锰佐剂背部及腹腔注射小鼠(50 µg/只);第二周和第四周进行第2次和第3次相同剂量免疫,3天后取脾细胞进行融合。复苏SP20骨髓瘤细胞,培养至其处于对数生长期。取免疫的BALB/c小鼠,摘除眼球采血供阳性对照血清,同时颈脱位处死小鼠,用75%酒精消毒体表3-5 min,取其脾脏,制备脾细胞悬浮液。
取上述脾细胞与骨髓瘤细胞按照5:1的比例,在无血清的1640培养基中混匀,1200rpm离心5分钟,充分吸取上清,轻轻震荡离心管底部,振散细胞,在45-60秒内加入1 mL预热过的50% PEG融合细胞,边加边轻轻摇匀,加完后静置90秒,加入无血清的1640培养基终止融合(第一分钟加1 mL,第二分钟加2 mL,第三分钟加8 mL),37℃静置10 min,1200 rpm离心5分钟,沉淀用HAT培养基悬浮,分装到96孔含有饲养细胞的细胞板中,37℃、5% CO2的细胞培养箱中培养。细胞培养箱中培养5天后,用HAT培养基换液一次,第10天用HAT培养基换液,等到融合细胞覆盖孔底10%-50%时,采用间接ELISA法筛选阳性克隆,保存克隆株,定名为小鼠杂交瘤细胞株4C9B12D7D12,保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏中心登记入册编号为CGMCC No. 45660,保藏日期为:2023年08月17日。
实施例3 单克隆抗体制备及其亚型分析
4C9B12D7D12杂交瘤细胞,用含10%胎牛血清的1640培养基培养。每只BALB/c雄性小鼠腹腔注射0.5 mL液体石蜡。10天后收集细胞,用10 mL生理盐水重悬细胞,每只小鼠腹腔注射0.5 mL(细胞密度大约为1×107个/mL)。2周后,收集腹水。以Thermo公司Melon GelMonoclonal IgG Purification Kit试剂盒进行抗体纯化,纯化后的抗体分装后于-20℃保存。
采用Pierce Papid Isotyping Kit-Mouse试剂盒抗体亚型鉴定,首先用样本稀释液将抗体稀释为500 ng/mL,然后每孔加入150 μL稀释好的抗体,10 min后观察记录结果。结果显示4C9B12D7D12单克隆抗体为小鼠IgG1亚型,如图2所示。
实施例4 抗人MMP19蛋白单克隆抗体可变区序列测定
培养小鼠杂交瘤细胞株4C9B12D7D12,Trizol法提取杂交瘤细胞总RNA,ThermoFisher公司High Capacity cDNA Rever Transcription Kit试剂盒逆转录cDNA后,根据《重组抗体》(科学出版社,沈倍奋主编,2005年出版)中鼠单抗引物序列,设计并由北京擎科生物科技有限公司合成该抗体的重轻链引物,PCR扩增(扩增程序为:95℃预热3 min,进行30个循环(95℃ 30秒,60℃ 30秒,72℃ 60秒),最后72℃延伸10 min),连接PMD18-T载体,转入大肠杆菌JM109中,挑选阳性克隆进行测序。将测定序列在BLAST网站(https://www.ncbi.nlm.nih.gov/igblast/)比对分析小鼠来源单克隆抗体CDR区序列。
经序列分析后发现,4C9B12D7D12单克隆抗体轻链可变区氨基酸序列为106个氨基酸,其序列如下:
IVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWK(SEQ ID NO:1),其中带有下划线的序列依次为LCDR1、LCDR2和LCDR3,其中,LCDR1位于26-35aa,氨基酸序列为KSVSTSGYSY(SEQ ID NO:2);LCDR2位于53-58aa,氨基酸序列为LVS;LCDR3位于92-99aa,氨基酸序列为QHIRELTR(SEQID NO:3)。
经序列分析后发现,4C9B12D7D12单克隆抗体重链可变区氨基酸序列为119个氨基酸,其序列如下:
QVQLKESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYISYSGSTRYNPSLISRVSITRDTSKNQFFLQLKSVTTEDTATYYCAKEGFDSTYAWFAYWGQGTLVTV(SEQ ID NO:4),其中带有下划线的序列依次为LCDR1、LCDR2和LCDR3,其中,LCDR1位于26-34aa,氨基酸序列为GYSITSDYA(SEQ ID NO:5);LCDR2位于52-58aa,氨基酸序列为ISYSGST(SEQ ID NO:6);LCDR3位于97-110aa,氨基酸序列为AKEGFDSTYAWFAY(SEQ ID NO:7)。
实施例5 抗人MMP19蛋白单克隆抗体活性测定
1、酶联免疫法
用碳酸盐包被缓冲液将MMP19蛋白稀释成浓度为2.5 μg/mL,每孔包被100 μL,4℃过夜;用洗液洗板2次,每孔300 μL;每孔加入120 μL封闭液室温封闭6小时;用洗液洗板5次,每孔300 μL;用抗体稀释液将4C9B12D7D12单克隆抗体进行梯度稀释,稀释比例为1:1000、1:3000、1:9000、1:27000、1:81000、1:243000和1:729000,以每孔100 μL加样并设置空白孔(100 μL抗体稀释液);37℃孵育30 min;用洗液洗板5次,每孔300 μL;HRP标记的山羊抗小鼠二抗37℃孵育20 min;用洗液洗板5次,每孔200 μL;加新鲜配制的底物溶液,每孔100 μL,37℃孵育10分钟;每孔加入50 μL 2M H2SO4终止反应,采用酶标仪波长450 nm测定各孔的吸光值,结果如表1所示,结果显示,所制备的单克隆抗体可以特异性识别人MMP19蛋白,并且所制备的单克隆抗体的效价大于1:243000,表明本发明制备的单克隆抗体对MMP19蛋白具有非常高的亲和力。
表1 小鼠抗人MMP19蛋白单克隆抗体的效价测定(OD值)
2、Western Blot免疫印迹法
将HEK293T细胞按每孔5×105的比例接种于6孔板中,铺制2个孔,次日,待细胞生长至80%左右(约12-16 h)时,在每个孔中采用jetPRIME试剂将1 µg pcDNA3.4/MMP19质粒和空载体转染HEK293T细胞。培养24 h后收集细胞,预冷的PBS洗2次,加入Thermo公司M-PERMammalian Protein Extraction Reagent裂解液冰上裂解细胞5 min,4℃ 16200×g离心15 min,收集上清,加入等体积的2×SDS上样缓冲液,沸水浴煮样5 min后,10% SDS-PAGE,转印至PVDF膜,5%脱脂奶粉室温封闭1 h,加入浓度为2 µg/mL的4C9B12D7D12单克隆抗体,4℃孵育过夜;0.1% TBST洗3次后,加入羊抗鼠-HRP抗体,室温孵育1 h,0.1% TBST洗3次后,ECL化学发光液显色,保存结果,结果如图3所示,结果显示,本发明制备的抗体能够特异性识别外源的MMP19蛋白。
3、双抗体夹心法检测MMP19抗原
用碳酸盐包被缓冲液将4C9B12D7D12单克隆抗体稀释成浓度为5 μg/mL,每孔包被100 μL,4℃过夜;用洗液洗板2次,每孔300 μL;每孔加入120 μL封闭液室温封闭6小时;每孔加入MMP19全长蛋白100 μL,浓度分别为900、300、100、33.3、11.1和0 ng/mL,37℃孵育30min;用洗液洗板5次,每孔300 μL;加入HRP标记的4C9B12D7D12抗体作为检测抗体,37℃孵育30 min;洗液洗板5次,加入TMB底物,37℃显色10 min;加入终止液50 μL,采用酶标仪波长450 nm测定各孔的吸光值。结果如表2所示,结果显示,本发明制备的抗体能够特异性识别MMP19抗原。
表2 双抗体夹心法检测MMP19抗原实验结果
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出,对于本领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以对本发明进行若干改进和修饰,这些改进和修饰也将落入本发明权利要求的保护范围内。
Claims (21)
1.一种抗MMP19蛋白的抗体,其特征在于,所述抗体包含重链可变区和轻链可变区;
所述重链可变区中HCDR1、HCDR2、HCDR3的氨基酸序列分别如SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7所示;
所述轻链可变区中LCDR1、LCDR3的氨基酸序列分别如SEQ ID NO:2、SEQ ID NO:3所示,所述轻链可变区中LCDR2的氨基酸序列为LVS。
2.一种核酸分子,其特征在于,所述核酸分子编码权利要求1所述的抗体。
3.一种表达载体,其特征在于,所述表达载体包含权利要求2所述的核酸分子。
4.一种宿主细胞,其特征在于,所述宿主细胞包含权利要求3所述的表达载体。
5.一株分泌权利要求1所述抗体的杂交瘤细胞株,其特征在于,所述杂交瘤细胞株于2023年08月17日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC NO. 45660。
6.一种用于检测MMP19蛋白的抗体偶联物,其特征在于,所述抗体偶联物为权利要求1所述的抗体直接或间接偶联至可检测标记物上形成的复合物。
7.一种检测试剂,其特征在于,所述检测试剂包含权利要求1所述的抗体、权利要求2所述的核酸分子、权利要求3所述的表达载体、权利要求4所述的宿主细胞、权利要求5所述的杂交瘤细胞株和/或权利要求6所述的抗体偶联物。
8.一种检测试剂盒,其特征在于,所述检测试剂盒包含权利要求1所述的抗体、权利要求2所述的核酸分子、权利要求3所述的表达载体、权利要求4所述的宿主细胞、权利要求5所述的杂交瘤细胞株、权利要求6所述的抗体偶联物和/或权利要求7所述的检测试剂。
9.一种检测试纸条,其特征在于,所述检测试纸条包含权利要求1所述的抗体。
10.一种生产权利要求1所述的抗体的方法,其特征在于,所述方法包括如下步骤:在适合的条件下,培养权利要求4所述的宿主细胞或权利要求5所述的杂交瘤细胞株,从宿主细胞培养产物中分离纯化得到权利要求1所述的抗体。
11.一种制备权利要求4所述的宿主细胞的方法,其特征在于,所述方法包括如下步骤:将权利要求2所述的核酸分子或权利要求3所述的表达载体导入到宿主细胞中,得到权利要求4所述的宿主细胞。
12.一种体外非治疗目的地抑制MMP19蛋白的方法,其特征在于,所述方法包括如下步骤:将权利要求1所述的抗体、权利要求2所述的核酸分子和/或权利要求3所述的表达载体导入到生物体细胞中,通过表达权利要求1所述的抗体抑制MMP19蛋白的活性。
13.一种非诊断和非治疗目的地检测MMP19蛋白的方法,其特征在于,所述方法包括如下步骤:将待测样品与权利要求1所述的抗体、权利要求6所述的抗体偶联物和/或权利要求7所述的检测试剂接触,检测MMP19蛋白与抗体免疫复合物的形成。
14.权利要求4所述的宿主细胞在制备权利要求1所述的抗体中的应用。
15.权利要求5所述的杂交瘤细胞株在制备权利要求1所述的抗体中的应用。
16.权利要求1所述的抗体、权利要求2所述的核酸分子、权利要求3所述的表达载体、权利要求4所述的宿主细胞和/或权利要求5所述的杂交瘤细胞株在制备用于检测MMP19蛋白的抗体偶联物中的应用。
17.权利要求1所述的抗体、权利要求2所述的核酸分子、权利要求3所述的表达载体、权利要求4所述的宿主细胞、权利要求5所述的杂交瘤细胞株和/或权利要求6所述的抗体偶联物在制备用于检测MMP19蛋白的检测试剂中的应用。
18.权利要求1所述的抗体、权利要求2所述的核酸分子、权利要求3所述的表达载体、权利要求4所述的宿主细胞、权利要求5所述的杂交瘤细胞株、权利要求6所述的抗体偶联物和/或权利要求7所述的检测试剂在制备用于检测MMP19蛋白的检测试剂盒中的应用。
19.权利要求1所述的抗体、权利要求2所述的核酸分子、权利要求3所述的表达载体、权利要求4所述的宿主细胞、权利要求5所述的杂交瘤细胞株、权利要求6所述的抗体偶联物和/或权利要求7所述的检测试剂在制备用于检测MMP19蛋白的检测试纸条中的应用。
20.权利要求1所述的抗体、权利要求2所述的核酸分子、权利要求3所述的表达载体、权利要求4所述的宿主细胞、权利要求5所述的杂交瘤细胞株、权利要求6所述的抗体偶联物、权利要求7所述的检测试剂、权利要求8所述的检测试剂盒和/或权利要求9所述的检测试纸条在制备用于诊断和/或辅助诊断MMP19蛋白相关疾病的产品中的应用。
21.根据权利要求20所述的应用,其特征在于,所述MMP19蛋白相关疾病包括结直肠癌、肺癌、胃癌、胰腺癌、甲状腺乳头状癌、卵巢癌、肝癌、骨肉瘤、胃癌、神经胶质瘤、类风湿关节炎、系统性红斑狼疮、多发性硬化症。
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101115836A (zh) * | 2005-02-09 | 2008-01-30 | 健泰科生物技术公司 | 用基质金属蛋白酶拮抗剂抑制her2脱落 |
CN112649608A (zh) * | 2020-12-01 | 2021-04-13 | 中国人民解放军军事科学院军事医学研究院 | 血清中mmp19在焦虑抑郁症中的应用 |
CN114774534A (zh) * | 2022-04-15 | 2022-07-22 | 苏州惟慎生物科技有限公司 | Mmp19作为肾脏早期损伤相关的生物标志物在早期诊断肾损伤中的应用 |
-
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- 2023-09-11 CN CN202311163159.0A patent/CN116925231B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101115836A (zh) * | 2005-02-09 | 2008-01-30 | 健泰科生物技术公司 | 用基质金属蛋白酶拮抗剂抑制her2脱落 |
CN112649608A (zh) * | 2020-12-01 | 2021-04-13 | 中国人民解放军军事科学院军事医学研究院 | 血清中mmp19在焦虑抑郁症中的应用 |
CN114774534A (zh) * | 2022-04-15 | 2022-07-22 | 苏州惟慎生物科技有限公司 | Mmp19作为肾脏早期损伤相关的生物标志物在早期诊断肾损伤中的应用 |
Non-Patent Citations (1)
Title |
---|
MMP19 is upregulated during melanoma progression and increases invasion of melanoma cells;Muller,M.等;Modern Pathology;511-521 * |
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