引用本文:	丛思璇,邓鹏辉,徐明策,闻建晴,王淏,毕允晨.Ⅱ型鲨鱼源单域抗体EF73在大肠杆菌中的稳定表达[J].海洋科学,2025,49(6):86-97.

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Ⅱ型鲨鱼源单域抗体EF73在大肠杆菌中的稳定表达
丛思璇^1,2, 邓鹏辉^1,2, 徐明策^1,2, 闻建晴^1,2, 王淏¹, 毕允晨^1,2,3
1.中国科学院海洋研究所实验海洋生物学重点实验室, 山东青岛 266000;2.中国科学院大学, 北京 100049;3.青岛海洋科技中心海洋生物学与生物技术功能实验室, 山东青岛 26600

摘要:

鲨鱼体内存在一种天然缺失轻链、仅含重链的重链抗体IgNAR (immunoglobulin new antigen receptor),其可变区VNAR (variable domain of new antigen receptor)能够独立结合抗原,与骆驼科重链抗体可变区VHH (variable domain of heavy chain of heavy-chain antibody)共同被称为单域抗体。稳定重组表达是鲨鱼源单域抗体VNAR研究和应用工作的重要前提,但VNAR表达过程中容易出现不同批次间表达产量差异大的问题。目前,有关鲨鱼源单域抗体重组表达的研究报道很少,本研究以鲨鱼源Ⅱ型VNAR EF73为研究对象,以在大肠杆菌中的稳定表达为目标优化表达策略。实验结果表明,采用多克隆表达替代常用的单克隆表达,以及将促溶标签TrxA与EF73融合表达等方式,可帮助EF73在大肠杆菌周质空间和胞质内两个区室均以可溶性蛋白形式表达,且批次间表现稳定。该研究为部分鲨鱼源单域抗体难以稳定表达的问题提出了解决方案,促进了鲨鱼源单域抗体的研究和应用。

关键词: 鲨鱼源单域抗体原核表达系统蛋白重组表达

DOI：10.11759/hykx20250430001

分类号:Q816

基金项目:国家自然科学基金(42306144); 青岛市关键技术攻关及产业化示范项目(25-1-1-gjgg-76-nsh)

Robust expression of the Type Ⅱ shark-derived single domain antibody EF73 in Escherichia coli

CONG Sixuan^1,2, DENG Penghui^1,2, XU Mingce^1,2, WEN Jianqing^1,2, WANG Hao¹, BI Yunchen^1,2,3

1.Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266000, China;2.University of Chinese Academy of Sciences, Beijing 100049, China;3.Laboratory for Marine Biology and Biotechnology, Qingdao Marine Science and Technology Center, Qingdao 266000, China

Abstract:

Sharks produce a heavy-chain antibody known as immunoglobulin new antigen receptor (IgNAR), which lacks light chains. Its variable region, the variable domain of the new antigen receptor (VNAR), can independently bind to antigens. Together with variable domain of heavy-chain antibodies (VHHs), VNARs are referred to as single-domain antibodies. Robust expression of shark recombinant VNARs is crucial for their characterization and application. However, their expression and yield often suffer from significant batch-to-batch variability, and reports on the recombinant VNAR expression remain limited. In this study, we devised a strategy for optimizing the expression of EF73, a Type Ⅱ shark-derived VNAR. Results showed that inoculating the initial culture with multiple colonies rather than a single colony, along with expressing EF73 as a TrxA fusion, consistently facilitated high-level expression of soluble EF73 in both the periplasm and cytoplasm. This strategy provides a practical approach to overcoming the challenges associated with VNAR expression and may promote further research and application of shark-derived single-domain antibodies.

Key words: variable domain of the new antigen receptor prokaryotic expression system recombinant protein expression