Expression, purification, and functional analysis of an antigen-targeting fusion protein composed of CD40 ligand and the C-terminal fragment of ovalbumin
Expression, purification, and functional analysis of an antigen-targeting fusion protein composed of CD40 ligand and the C-terminal fragment of ovalbumin
复制标题
DOI：
10.1016/j.pep.2017.09.015
复制
发表时间：
2018-02-01
影响因子：
1.6
通讯作者：
Lee, Song F.
中科院分区：
文献类型：
--
作者：
Shi, Yunnuo;Halperin, Scott A.;Lee, Song F.
通过针对抗原提呈细胞表面受体的分子传递抗原是提高免疫反应的一种策略。本研究通过基因融合的方法构建了由卵清蛋白C端片段和小鼠CD40配体胞外区组成的抗原靶向融合蛋白(OVA-CD40LS)。在大肠杆菌中克隆了OVA-CD40LS和对照OVA(RoVa)基因，并以不溶性蛋白的形式高效表达。用镍亲和层析从大肠杆菌细胞裂解液中分离纯化RoVa蛋白，再经分步透析复性，得率为11.8 mg/L。OVA-CD40LS通过镍亲和层析和柱上蛋白质复性层析得到纯化。产量为528g/L。纯化的OVA-CD40LS，而不是RoVa，能够模拟小鼠骨髓来源的树突状细胞产生促炎细胞因子并上调细胞表面标志蛋白。纯化的OVA-CD40LS经小鼠口腔黏膜下注射后，可产生较强的免疫应答。总之，结果表明OVA-CD40LS融合蛋白具有生物学活性，具有抗原靶向蛋白的功能。(C)2017 Elsevier Inc.保留所有权利。
Delivering antigen via molecules specifically targeting receptors on the surface of antigen-presenting cells is a strategy to improve immune responses. In this study, an antigen-targeting fusion protein (OVA-CD40LS) composed of the C-terminal fragment of ovalbumin and the extracellular domain of mouse CD40 ligand was constructed by genetic fusion. The OVA-CD40LS and the control OVA (rOVA) genes were cloned in Escherichia coli and over-expressed as insoluble proteins. The rOVA protein was purified from the insoluble fraction of E. coli cell lysate by nickel affinity chromatography and refolded by step-wise dialysis to give a yield of 11.8 mg/L of culture. The OVA-CD40LS was purified by a 'two-round' nickel affinity and on-column protein-refolding chromatography. The yield was 528 g/L of culture. The purified OVA-CD40LS, but not the rOVA, was able to simulate the production of pro-inflammatory cytokines and up-regulate cell surface marker proteins in mouse bone marrow-derived dendritic cells. The purified OVA-CD40LS elicited a robust immune response when injected submucosally in the oral cavity of mice. Collectively, the results indicate that the OVA-CD40LS fusion protein was biologically active, functioning as an antigen-targeting protein. (C) 2017 Elsevier Inc. All rights reserved.