[基于转谷氨酰胺酶的抗体-药物偶联:抗体位点特异性突变和鉴定]。

药学学报 Pub Date : 2017-03-01

Zhao-xiong Ma, Yao Xu, Hong Zhao, Fu-mou Sun, Xin-rong Zhang, Min Wang, Juan Zhang

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引用次数: 0

摘要

转谷氨酰胺酶(TG)抗体的翻译后修饰允许更精确的偶联。基于抗cd24抗体(cG7)的氨基酸序列，本文旨在生成一个去糖基化的cG7突变体(cG7Q)。首先，我们通过定点诱变在297(N297Q)位置添加谷氨酰胺，然后通过电穿孔法将重组质粒转染CHO-s细胞并进行Dot blot筛选。随后，通过蛋白A亲和层析表达和纯化cG7Q，并进一步通过SDS-PAGE电泳和Western blot进行鉴定。采用表面等离子体共振和流式细胞术检测其亲和力，乳酸脱氢酶(LDH)释放量检测其ADCC作用。最终，cG7突变体cG7Q被成功表达，其序列特异性偶联位点可供进一步研究。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

本刊更多论文

[Transglutaminase-based antibody-drug conjugation: antibody site-specific mutation and identification].

Transglutaminase (TG) posttranslational modification of antibody permits more precisely conjugating. Based on the amino acid sequence of an anti-CD24 antibody (cG7), this article is aimed to generate a deglycosylated cG7 mutant (cG7Q). Firstly, we introduced additional glutamines at position 297 (N297Q) by site-directed mutagenesis, and then transfected the recombinant plasmids into CHO-s cells via electroporation method and screened by Dot blot assay. Subsequently, cG7Q was expressed and purified through Protein A affinity chromatography, further identified by SDS-PAGE electrophoresis and Western blot. Its affinity was detected with surface plasmon resonance and flow cytometry assay, and ADCC effect was determined by lactate dehydrogenase (LDH) release. Eventually, a cG7 mutant, cG7Q was successfully expressed with sequence-specific conjugation sites for further study.

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来源期刊

药学学报 Pharmacology, Toxicology and Pharmaceutics-Pharmacology, Toxicology and Pharmaceutics (all)

CiteScore

1.20

自引率

0.00%

发文量

期刊介绍： Acta Pharmaceutica Sinica B (APSB) is a bimonthly English peer-reviewed online journal in ScienceDirect, which publishes significant original research articles, communications and high quality reviews of recent advances. APSB encourages submissions from all areas of pharmaceutical sciences, including pharmacology, pharmaceutics, medicinal chemistry, natural products, pharmacognosy, pharmaceutical analysis and pharmacokinetics. APSB is a part of the series Acta Pharmaceutica Sinica, which was founded in 1953. The journal is co-published by Elsevier B.V., in association with the Institute of MateriaMedica, Chinese Academy of Medical Sciences and Chinese Pharmaceutical Association.