CN111100202B - 针对蛋白标签Streptactin的人源单克隆抗体 - Google Patents

针对蛋白标签Streptactin的人源单克隆抗体 Download PDF

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CN111100202B
CN111100202B CN201911422488.6A CN201911422488A CN111100202B CN 111100202 B CN111100202 B CN 111100202B CN 201911422488 A CN201911422488 A CN 201911422488A CN 111100202 B CN111100202 B CN 111100202B
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龚睿
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Wuhan Banke Biotechnology Co ltd
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1275—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Streptococcus (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/005—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

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Abstract

本发明公开了一种针对蛋白标签Streptactin的人源单克隆抗体。通过筛选和基因改造后，所获抗体的特异性好、亲和力高、抗聚集性增强，适用于带有该标签的目的蛋白的纯化和检测。

Description

针对蛋白标签Streptactin的人源单克隆抗体
技术领域
本发明涉及生物技术领域，具体地指一种针对蛋白标签Streptactin的人源单克隆抗体。
背景技术
Streptactin(氨基酸序列：WSHPQFEK(GGGS)2GGSAWSHPQFEK)是一种常用的蛋白纯化和检测的标签，可加在目的蛋白的N末端或者C末端，用于目的蛋白的纯化和检测。目前已有的针对该序列的抗体均为动物来源的，但在检测方面存在特异性不好等问题，这里，我们利用人源Fab噬菌体展示文库，筛选到针对该标签的人源单克隆抗体，可用于含有该标签的蛋白的纯化和检测，提高检测的特异性等。
发明内容
本发明的目的在于克服上述缺陷，提供一种针对蛋白标签Streptactin的人源单克隆抗体。
为实现上述目的，本发明提供了一种针对蛋白标签Streptactin的人源单克隆抗体，其轻链序列如SEQ No.1所示，其重链序列如SEQ No.3所示。
上述针对蛋白标签Streptactin的人源单克隆抗体对应的碱基序列，其轻链序列如SEQ No.4所示，其重链序列如SEQ No.5所示。
本发明的有益效果：通过筛选和基因改造后，所获抗体的特异性好、亲和力高、抗聚集性增强，适用于带有该标签的目的蛋白的纯化和检测。
附图说明
图1-1为Tango对A1抗体氨基酸序列的易聚集区预测。
图1-2为Tango对突变后A8氨基酸序列的易聚集区预测。
图1-3为A1和A8的重链突变区域比较图。
图2为SDS-PAGE检测A8抗体图。
图3为ELISA分析A8抗体与含有streptactin标签的目的蛋白的结合图。
具体实施方式
以下结合附图和具体实施例对本发明作进一步的详细描述。以下实施例是在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护范围不限于下述的实施例。
实施例1：A1抗体序列的获得
按照已有的文献(Zhu Z,Dimitrov DS,Methods Mol Biol,2009)构建了人源Fab噬菌体展示文库，并基于该文库对含有Streptactin标签的蛋白进行了三轮的筛选，并通过单克隆phage ELISA，将其中的阳性克隆送测序，得到了A1候选克隆的碱基序列，并经氨基酸翻译软件(https://web.expasy.org/translate/)，得到了其氨基酸序列，轻链氨基酸序列如SEQ No.1所示，其重链氨基酸序列如SEQ No.2所示。
实施例2：利用Tango软件对获得的A1氨基酸序列进行优化
利用Tango软件(http://tango.crg.es/)对A1候选克隆的氨基酸序列进行易聚集区预测，发现其中存在较强的易聚集区域，如图1-1，因此对该区域进行点突变，得到A8抗体，其轻链氨基酸序列如SEQ No.1所示，其重链氨基酸序列如SEQ No.3所示，对应碱基序列，轻链如SEQ No.4所示，重链如SEQ No.5所示。突变后的氨基酸序列经Tango软件重新预测，发现该易聚集区被明显改善，如图1-2，1-3。
实施例3：293F细胞表达A8抗体并SDS-PAGE检测
将突变后的碱基序列克隆到双启动载体pVitro2-neo-mcs(InvivoGen)上，利用PEI转染293F细胞后进行蛋白的表达，经过5-7天的表达，将293F细胞的培养上清经ProteinA(GE)纯化，将纯化后的蛋白经过浓换液后，得到溶于PBS缓冲液的A8抗体。经SDS-PAGE电泳检测，发现其呈现轻链和重链两条带，如图2所示。
实施例4：ELISA分析A8抗体与含有Streptactin标签的蛋白的结合
包被目的蛋白在ELISA板上，将A8抗体经梯度稀释后作为一抗，HRP标记的Goat-anti-human IgG-Fc(Abcam)作为二抗，显色后，通过信号强度变化计算EC50，约在100nM，如图3所示。
SEQUENCE LISTING
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tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct 1320
ccgggtaaa 1329

Claims (2)

1.一种针对蛋白标签Streptactin的人源单克隆抗体，其特征在于，其轻链序列如SEQNo.1所示，其重链序列如SEQ No.3所示。
2.根据权利要求1所述针对蛋白标签Streptactin的人源单克隆抗体，其特征在于，所述轻链的编码核苷酸序列如SEQ No.4所示，所述重链的编码核苷酸序列如SEQ No.5所示。
CN201911422488.6A 2019-12-31 2019-12-31 针对蛋白标签Streptactin的人源单克隆抗体 Active CN111100202B (zh)

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CN104437411A (zh) * 2014-12-15 2015-03-25 武汉大学 可逆组装和分解的Strep-tag多肽标记的生物分子衍生化基质及其应用
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EP2871189A1 (en) * 2013-11-07 2015-05-13 Institut Pasteur High-affinity monoclonal anti-strep-tag antibody
CN104437411A (zh) * 2014-12-15 2015-03-25 武汉大学 可逆组装和分解的Strep-tag多肽标记的生物分子衍生化基质及其应用

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