CN117603352B - 一种ctla-4蛋白纳米抗体 - Google Patents
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Abstract
本发明提供一种CTLA‑4蛋白纳米抗体,其中抗CTLA‑4蛋白纳米抗体氨基酸序列为SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3,抗CTLA4_2B1蛋白纳米抗体核酸序列为SEQ ID NO:4,抗CTLA4_2A4蛋白纳米抗体核酸序列为SEQ ID NO:5,抗CTLA4_1H11蛋白纳米抗体核酸序列为SEQ ID NO:6。本发明公开的CTLA‑4蛋白纳米抗体以及氨基酸序列、核酸序列具有高稳定性,耐酸碱pH,耐高温,能避免常规抗体需要低温储存和运输的要求,有利于大规模普及应用;且VHH序列与人VH序列同源性高,少数氨基酸突变即可以实现单域抗体的人源化;量产成本较低,易于大规模重组制备。
Description
技术领域
本发明涉及生物技术领域,具体为一种CTLA-4蛋白纳米抗体。
背景技术
CD28和其配体B7-1(CD80)/B7-2(CD86)对T细胞激活起作用,这与其中发挥反馈抑制调节的肿瘤免疫靶点CTLA-4(CD152)一起构成了最明确的调节T细胞途径。在研究肿瘤癌症的过程中,纳米抗体药物在疾病体外诊断和疾病治疗中具有重要作用。近期研究表明,国内外CTLA-4开发主要分两种治疗方式,单抗或和PD-1/PD-L1单抗联用;关联其他热门靶点构建双特异性抗体。
现有的主要技术方案是:先制备CTLA-4蛋白抗原,将抗原注射至大鼠或小鼠体内,依靠动物的免疫系统产生抗体;采集动物的血液并分离、提取出血清,再进一步分离出含有特异性抗体的成分,但是该基于鼠等动物的免疫系统产生肿瘤免疫靶点CTLA-4的抗体并分离提取的技术,有以下缺点:(1)抗体的特异性有限。(2)抗体的稳定性较差。(3)规模化批量生产成品高。且操作复杂、成本昂贵,不利于规模化批量生产。
发明内容
针对现有技术存在的不足,本发明目的是提供一种CTLA-4蛋白纳米抗体,以解决上述背景技术中提出的问题,本发明提供包括CTLA-4蛋白纳米抗体氨基酸序列和抗CTLA-4蛋白纳米抗体核酸序列。
为了实现上述目的,本发明是通过如下的技术方案来实现:一种CTLA-4蛋白纳米抗体,所述抗体序列包括:
编码抗CTLA4_2B1蛋白纳米抗体氨基酸序列为SEQ ID NO:1;编码抗CTLA4_2A4蛋白纳米抗体氨基酸序列为SEQ ID NO:2;编码抗CTLA4_1H11蛋白纳米抗体氨基酸序列为SEQID NO:3;编码抗CTLA4_2B1蛋白纳米抗体核酸序列为SEQ ID NO:4;编码抗CTLA4_2A4蛋白纳米抗体核酸序列为SEQ ID NO:5;编码抗CTLA4_1H11蛋白纳米抗体核酸序列为SEQ IDNO:6。
进一步的,该序列的筛选过程包括以下几个阶段:羊驼免疫注射、构建羊驼的纳米抗体文库、噬菌体表面展示筛选特异性纳米抗体,所述羊驼免疫注射过程中,采集大量的羊驼静脉外周血液,便于在后续筛选时得到高度多样性的纳米抗体;所述构建羊驼的纳米抗体文库时,采集的2批次羊驼静脉外周血液为原料进行构建高多样性的纳米抗体文库,且2批次羊驼静脉外周血液的处理方法相同;所述噬菌体表面展示筛选特异性纳米抗体将所述纳米抗体文库为来源,经噬菌体表面展示筛选得到抗原特异性的纳米抗体。
所述羊驼免疫注射包括以下步骤:将制备好的抗原平均分装成4份;累计对羊驼进行4次免疫,将抗原经皮下注射至动物体内。记第一次免疫为第一天,后续的免疫分别于第10天、第19天、第28天。
进一步的,第28天,于第四次免疫注射前,采集约200mL羊驼静脉外周血液;第42天,即第四次免疫之后14天,采集约300mL羊驼静脉外周血液。
进一步的,所述构建羊驼的纳米抗体文库具体步骤如下:使用密度梯度离心等方法,从羊驼静脉外周血液中分离得到淋巴细胞;提取淋巴细胞的总mRNA,并反转录为cDNA。
进一步的,使用适当的DNA引物,以上述cDNA为模板,经聚合酶链式反应(PCR)扩增得到羊驼免疫球蛋白IgG2和IgG3的VHH片段,即纳米抗体的DNA片段。
进一步的,将VHH的DNA连接至噬菌体表面展示筛选载体,构成VHH-pIII融合蛋白表达载体质粒库。其中,pIII是存在于噬菌体表面鞭毛上的蛋白质;将DNA连接产物经电转化方法,转化至TG1感受态细菌,适当培养后收集全部菌落,即为羊驼的纳米抗体文库。
进一步的,所述噬菌体表面展示筛选特异性纳米抗体过程如下:取适量冻存的纳米抗体文库,接种至细菌培养基,经适当培养后加入适量的辅助噬菌体,继续于适量条件下培养;以PEG-NaC法提取细菌培养上清中扩增的噬菌体。
进一步的,将噬菌体于抗原适当孵育,若抗原是细胞膜蛋白,可取噬菌体与富集的过表达抗原的细胞整体或细胞膜提取物孵育;若抗原是细胞内蛋白或分泌蛋白,可预先将抗原固定于试管、微孔板等介质,再与噬菌体孵育;淘洗,弃去噬菌体,再以适当的缓冲液润洗抗原适当次数,淘洗、除去与抗原非特异性结合的噬菌体,保留与抗原特异性结合的噬菌体;洗脱,以合适的方法处理与抗原特异性结合的噬菌体(如酸性甘氨酸溶液等),使噬菌体与抗原解离并保留,最终获取表达有特异性纳米抗体的噬菌体。
进一步的,将所述表达有特异性纳米抗体的噬菌体转变为特异性的纳米抗体文库,将噬菌体再次侵染培养至合适状态的大肠杆菌,但不再加入辅助噬菌体,待噬菌体侵染完全后,特异性的纳米抗体即以DNA质粒的形式存在于大肠杆菌中。
进一步的,取少量噬菌体稀释后再次侵染培养至合适状态的大肠杆菌,但不再加入辅助噬菌体,待噬菌体侵染完全后,将这些大肠杆菌均匀涂抹于细菌培养皿得到含有纳米抗体DNA质粒的单克隆菌落。
本发明的有益效果:
1.该CTLA-4蛋白纳米抗体特异性强,亲和力高。本发明得到的纳米抗体,其kd值分别为118.8nM、194.6nM和158.4nM,亲和力高,且特异性识别并结合CTLA4蛋白;有潜在疾病诊断和治疗价值。所涉及的抗CTLA-4纳米抗体有望做介导Treg耗尽或功能阻断,从而增强T细胞激活,杀死肿瘤细胞,增强应对癌症的免疫反应。
2.该CTLA-4蛋白纳米抗体序列相对分子质量小,可溶性高,组织渗透性强,分布性好,扩散和代谢迅速,有望对癌症诊断、抗癌药物的开发等相关应用做出巨大贡献;且结构简单,容易进行基因工程改造,具有成熟的优化策略用于增强单域抗体亲和力、延长体内半衰期以及与其它分子偶联用于药物开发,如连接放射性同位素,偶联传递药物、CART和荧光标记高分辨成像等。
3.该CTLA-4蛋白纳米抗体人源化更简单。VHH序列与人VH序列同源性高,少数氨基酸突变即可以实现单域抗体的人源化;具有高稳定性,耐酸碱pH,耐高温,能避免常规抗体需要低温储存和运输的要求,有利于大规模普及应用;量产成本较低,易于大规模重组制备。
附图说明
图1为本发明实施例中抗CTLA-4蛋白纳米抗体第一组实验数据图;
图2为本发明实施例中抗CTLA-4蛋白纳米抗体第二组实验数据图;
图3为本发明实施例中抗CTLA-4蛋白纳米抗体第三组实验数据图。
具体实施方式
为使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解,下面结合具体实施方式,进一步阐述本发明。
请参阅图1至图3,本发明提供一种技术方案:一种CTLA-4蛋白纳米抗体,其中抗CTLA-4蛋白纳米抗体氨基酸序列、抗CTLA-4蛋白纳米抗体核酸序列如下表所示,其中编码抗CTLA4_2B1蛋白纳米抗体氨基酸序列为SEQ ID NO:1;编码抗CTLA4_2A4蛋白纳米抗体氨基酸序列为SEQ ID NO:2;编码抗CTLA4_1H11蛋白纳米抗体氨基酸序列为SEQ ID NO:3;编码抗CTLA4_2B1蛋白纳米抗体核酸序列为SEQ ID NO:4;编码抗CTLA4_2A4蛋白纳米抗体核酸序列为SEQ ID NO:5;编码抗CTLA4_1H11蛋白纳米抗体核酸序列为SEQ ID NO:6。
序列表:
SEQ ID NO:1
QVQLQESGGGLVQAGDSLRLSCAASGRTSRRHAMGWFRQSPGKEREFVAGVSWGDSTVYADSVKGRFAISRDNAKNTLYLQMNSLKPEDTAVYYCFADVRTTSWGPSRRYWGQGTQVTVSS
SEQ ID NO:2
EVQLVESGGGFVQAGGSLRVSCAASGRTFSSYTMGWFRQAPGKEREFVAGITRNGRSTYYADSVKGRFTISRDNAKNTGYLQMNSLKPEDTAVYYCFADVRTTNWGPLRRYWGQGTQVTVSS
SEQ ID NO:3
QVQLVQSGGGLVQPGGSLRLSCAASGRTIWRRTMGWFRQAPGKEREFVAAIISGTTYYADSVKGRFTISRDNAKNTVYLHMNNLKPDDTAVYYCAAESALKTKRHPSWVFQRDEHTYWGQGTQVTVSS
SEQ ID NO:4
CAGGTGCAGCTGCAGGAGTCGGGGGGAGGATTGGTGCAGGCTGGAGACTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCAGCCGTCGCCATGCCATGGGCTGGTTCCGCCAAAGTCCAGGGAAGGAGCGTGAGTTTGTAGCAGGTGTTAGCTGGGGTGATAGTACTGTCTATGCAGACTCCGTGAAGGGCCGATTCGCCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTACAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTTTCGCAGATGTGCGTACTACAAGTTGGGGGCCGTCCCGGCGCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO:5
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGATTTGTGCAGGCTGGGGGCTCTCTGAGAGTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTAGCTATACCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAATTTGTCGCAGGTATTACGAGGAATGGTCGGAGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGGGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTTTCGCAGATGTGCGTACTACAAATTGGGGGCCGCTCCGGCGCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
SEQ ID NO:6
CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCATCTGGCGCAGAACCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAATTTGTAGCAGCAATTATTAGTGGTACCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAATACGGTGTATCTGCATATGAACAACCTGAAACCTGACGACACGGCCGTTTATTACTGTGCAGCGGAATCAGCCCTGAAGACTAAACGCCACCCCTCATGGGTATTCCAGAGGGACGAGCACACCTACTGGGGCCAGGGGACCCAGGTCACTGTCTCCTCA
具体制备方法分为如下几个阶段:
1.羊驼免疫注射
本实施例,不同于传统技术依赖于鼠、兔、猴、羊等经典的模式动物,本发明的技术方案是依靠羊驼的免疫系统产生的抗体,被成为“纳米抗体”。纳米抗体是从骆驼、鲨鱼等动物体内免疫球蛋白分离出的微小抗体片段,它具有与完整抗体相同的抗原结合能力和结构稳定性,是现有可结合目标抗原的最小的单位,相对分子质量仅为15kD。相比于传统的鼠、兔等动物仅能识别抗原表面平展的多肽,羊驼等动物体内的免疫系统能够识别抗原表面复杂的空间结构,能够产生高度特异性、高亲和力的纳米抗体。将制备好的抗原平均分装成4份,具体的技术方案是:
1.1累计对羊驼进行4次免疫,将抗原经皮下注射至动物体内。记第一次免疫为第一天,后续的免疫分别于第10天、第19天、第28天;
1.2第28天,于第四次免疫注射前,采集约200mL羊驼静脉外周血液;
1.3第42天,即第四次免疫之后14天,采集约300mL羊驼静脉外周血液。
相比于传统鼠、兔等动物抗体的免疫技术方案,本技术优势在于采集大量的羊驼静脉外周血液,有利于后续筛选得到高度多样性的纳米抗体。
2.构建羊驼的纳米抗体文库
本实施例,以第一步中采集的2批次羊驼静脉外周血液为原料,构建高多样性的纳米抗体文库。2批次羊驼静脉外周血液的处理方法相同,具体技术方案是:
2.1使用密度梯度离心等方法,从羊驼静脉外周血液中分离得到淋巴细胞;
2.2提取淋巴细胞的总mRNA,并反转录为cDNA;
2.3使用适当的DNA引物,以上述cDNA为模板,经聚合酶链式反应(PCR)扩增得到羊驼免疫球蛋白IgG2和IgG3的VHH片段,即纳米抗体的DNA片段;
2.4将VHH的DNA连接至噬菌体表面展示筛选载体,构成VHH-pIII融合蛋白表达载体质粒库。其中,pIII是存在于噬菌体表面鞭毛上的蛋白质。
2.5将DNA连接产物经电转化方法,转化至TG1感受态细菌,适当培养后收集全部菌落,即为羊驼的纳米抗体文库。
相比于传统的从鼠、兔等动物血清或淋巴细胞中分离得到抗体的方法,本技术方案能够长期保存持有羊驼的全部纳米抗体片段(即文库),能够持续地支撑后续不断地进行纳米抗体的筛选与开发。
3.噬菌体表面展示筛选特异性纳米抗体
本实施例,以第二步得到的纳米抗体文库为来源,经噬菌体表面展示筛选得到抗原特异性的纳米抗体。具体技术方案如下:
3.1取适量冻存的纳米抗体文库,接种至细菌培养基,经适当培养后加入适量的辅助噬菌体,继续于适量条件下培养;
3.2以PEG-NaC法提取细菌培养上清中扩增的噬菌体;
3.3将噬菌体于抗原适当孵育。若抗原是细胞膜蛋白,可取噬菌体与富集的过表达抗原的细胞整体或细胞膜提取物孵育;若抗原是细胞内蛋白或分泌蛋白,可预先将抗原固定于试管、微孔板等介质,再与噬菌体孵育。
3.4淘洗。弃去噬菌体,再以适当的缓冲液(如PBS等)润洗抗原适当次数,淘洗、除去与抗原非特异性结合的噬菌体,保留与抗原特异性结合的噬菌体。
3.5洗脱。以合适的方法处理与抗原特异性结合的噬菌体(如酸性甘氨酸溶液等),使噬菌体与抗原解离并保留。至此,即得到了表达有特异性纳米抗体的噬菌体,这些噬菌体可进行下述技术操作:
3.6转变为特异性的纳米抗体文库。将噬菌体再次侵染培养至合适状态的大肠杆菌,但不再加入辅助噬菌体,待噬菌体侵染完全后,特异性的纳米抗体即以DNA质粒的形式存在于大肠杆菌中。收集这些全部的大肠杆菌,即成为抗原特异性的纳米抗体文库,可以此文库为原料,返回步骤(1)进行下一轮的噬菌体表面展示筛选;
本实施例,转变为单克隆纳米抗体菌落。取少量步骤3.5中得到的噬菌体(如0.5%),稀释后再次侵染培养至合适状态的大肠杆菌,但不再加入辅助噬菌体,待噬菌体侵染完全后,将这些大肠杆菌均匀涂抹于细菌培养皿,适当调节下培养即可得到含有纳米抗体DNA质粒的单克隆菌落。
以上显示和描述了本发明的基本原理和主要特征和本发明的优点,对于本领域技术人员而言,显然本发明不限于上述示范性实施例的细节,而且在不背离本发明的精神或基本特征的情况下,能够以其他的具体形式实现本发明。因此,无论从哪一点来看,均应将实施例看作是示范性的,而且是非限制性的,本发明的范围由所附权利要求而不是上述说明限定,因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。不应将权利要求中的任何附图标记视为限制所涉及的权利要求。
此外,应当理解,虽然本说明书按照实施方式加以描述,但并非每个实施方式仅包含一个独立的技术方案,说明书的这种叙述方式仅仅是为清楚起见,本领域技术人员应当将说明书作为一个整体,各实施例中的技术方案也可以经适当组合,形成本领域技术人员可以理解的其他实施方式。
Claims (4)
1.一种CTLA-4蛋白纳米抗体,其特征在于,基于羊驼体内免疫球蛋白分离出的微小抗体片段,该抗CTLA-4蛋白纳米抗体氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2、SEQ IDNO:3所示。
2.一种核酸分子,其特征在于,所述核酸分子编码CTLA4_2B1纳米抗体,所述核酸分子核酸序列为SEQ ID NO:4。
3.一种核酸分子,其特征在于,所述核酸分子编码CTLA4_2A4纳米抗体,所述核酸分子核酸序列为SEQ ID NO:5。
4.一种核酸分子,其特征在于,所述核酸分子编码CTLA4_1H11纳米抗体,所述核酸分子核酸序列为SEQ ID NO:6。
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