靶向VEGFA结合蛋白的设计与功能表征

doi:10.3969/j.issn.1674-8115.2026.09.006

上海交通大学学报（医学版） ›› 2026, Vol. 46 ›› Issue (9): 1212-1221.doi: 10.3969/j.issn.1674-8115.2026.09.006

• 论著 · 基础研究 • 上一篇

靶向VEGFA结合蛋白的设计与功能表征

卢悦¹^,², 颜伟康¹^,²^,³, 刘颖斌¹^,²^,³, 何勇宁¹^,²^,³()

^1.上海交通大学医学院附属仁济医院肿瘤系统医学全国重点实验室，上海市肿瘤研究所，上海 200127
^2.上海交通大学医学院附属仁济医院上海市肿瘤系统调控与转化重点实验室，上海 200127
^3.上海交通大学医学院附属仁济医院胆胰外科，上海 200127

收稿日期:2025-12-10 接受日期:2026-04-21 出版日期:2026-09-28 发布日期:2026-09-28
通讯作者: 何勇宁，研究员，博士；电子信箱：heyn@shsmu.edu.cn。
基金资助:
肿瘤系统医学全国重点实验室科研项目(ZZ-94-25-17)

Design and functional characterization of VEGFA-targeted binding proteins

Lu Yue¹^,², Yan Weikang¹^,²^,³, Liu Yingbin¹^,²^,³, He Yongning¹^,²^,³()

^1.State Key Laboratory of Systems Medicine for Cancer, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China
^2.Shanghai Key Laboratory for Cancer System Regulation and Clinical Translation (CSRCT-SHANGHAI), Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China
^3.Department of Biliary-Pancreatic Surgery, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China

Received:2025-12-10 Accepted:2026-04-21 Online:2026-09-28 Published:2026-09-28
Contact: He Yongning, E-mail: heyn@shsmu.edu.cn.
Supported by:
Research Project of State Key Laboratory of Systems Medicine for Cancer(ZZ-94-25-17)

摘要/Abstract

摘要：

目的·应用蛋白质从头设计策略构建靶向血管内皮生长因子A（vascular endothelial growth factor A，VEGFA）的结合蛋白，并探索该蛋白对VEGFA介导的人脐静脉内皮细胞（human umbilical vein endothelial cells，HUVECs）生物学效应的影响。方法·利用Rosetta RifDock、FastDesign、ProteinMPNN等多算法联合流程设计靶向VEGFA的结合蛋白，并采用AlphaFold预测模型评估该结合蛋白的结构合理性及其与VEGFA的相互作用。采用酵母表面展示技术结合双荧光激活细胞分选，筛选靶向VEGFA的特异性结合蛋白，再经大肠埃希菌表达系统进行表达与纯化。借助生物膜干涉（bio-layer interferometry，BLI）技术测定结合蛋白与VEGFA的亲和力；联合细胞计数试剂盒-8（cell counting kit-8，CCK-8）实验、Annexin Ⅴ/PI双染法、蛋白质印迹法（Western blotting）、细胞划痕愈合实验、体外血管生成实验等HUVECs相关细胞实验，探索该结合蛋白对VEGFA介导的HUVECs生物学效应的影响。结果·成功设计了靶向VEGFA的结合蛋白，并建立了候选结合蛋白库；经酵母表面展示技术筛选与BLI测定，获得了1个与VEGFA中等强度结合的结合蛋白-3（minibinder-3，MB3）。相关细胞实验的结果显示，MB3能够抑制VEGFA介导的HUVECs存活、抗凋亡、迁移与体外血管生成，并能够降低由VEGFA激活的血管内皮细胞生长因子受体（vascular endothelial growth factor receptor，VEGFR）下游信号分子蛋白激酶B（protein kinase B，AKT）及细胞外信号调节激酶（extracellular signal-regulated kinase，ERK）的磷酸化水平（均P<0.05）。结论·基于蛋白质从头设计流程，获得了中等亲和力的VEGFA结合蛋白MB3；体外实验证实其可抑制由VEGFA介导的HUVECs的功能及相关信号通路，同时验证了该设计流程在抗VEGFA人工蛋白研发中的可行性，为肿瘤血管靶向治疗提供了新思路。

关键词: 蛋白质从头设计, 血管内皮生长因子A, 结合蛋白, 抗血管生成治疗

Abstract:

Objective ·To construct vascular endothelial growth factor A (VEGFA)-targeted binding proteins by using a de novo protein design strategy, and verify the effects of these proteins on VEGFA-induced biological activities in human umbilical vein endothelial cells (HUVECs). Methods ·A multi-algorithm workflow integrating Rosetta RifDock, FastDesign, and ProteinMPNN was used to design VEGFA-targeted binding proteins. The structural plausibility of the binding proteins and their interactions with VEGFA were assessed using the AlphaFold prediction model. Yeast surface display combined with dual-fluorescence-activated cell sorting was then used to screen specific VEGFA-targeted binding proteins, which were subsequently expressed and purified using the E. coli expression system. The binding affinities between the binding proteins and VEGFA were measured using bio-layer interferometry (BLI). Finally, cell counting kit-8 (CCK-8) assays, Annexin Ⅴ/PI double-staining, Western blotting, scratch wound healing assays, and in vitro tube formation assays were performed to evaluate the effects of the binding protein on VEGFA-induced biological activities in HUVECs. Results ·VEGFA-targeted binding proteins were successfully designed and a candidate binding protein library was constructed. One protein, minibinder-3 (MB3), with moderate affinity for VEGFA was obtained after yeast surface display screening and BLI measurement. Subsequent in vitro experiments demonstrated that MB3 inhibited VEGFA-induced survival, resistance to apoptosis, migration, and angiogenesis, and also reduced the VEGFA-induced phosphorylation of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK), which are downstream signaling molecules of vascular endothelial growth factor receptor (VEGFR) (all P<0.05). Conclusion ·Based on the de novo protein design workflow, MB3, a VEGFA-binding protein with moderate affinity, was obtained. In vitro experiments confirmed that MB3 inhibited VEGFA-mediated HUVEC functions and related signaling pathways, thereby demonstrating the feasibility of the design workflow for developing anti-VEGFA artificial proteins and providing new insights into tumor vascular-targeted therapy.

Key words: de novo protein design, vascular endothelial growth factor A (VEGFA), binding protein, anti-angiogenic therapy

中图分类号:

卢悦, 颜伟康, 刘颖斌, 何勇宁. 靶向VEGFA结合蛋白的设计与功能表征[J]. 上海交通大学学报（医学版）, 2026, 46(9): 1212-1221.

Lu Yue, Yan Weikang, Liu Yingbin, He Yongning. Design and functional characterization of VEGFA-targeted binding proteins[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2026,(9): 1212-1221.

图/表 5

图1 VEGFA/VEGFR2复合物结构（A）及靶向VEGFA结合蛋白设计位点（B）的缎带图

Fig 1 Ribbon diagram of the VEGFA/VEGFR2 complex structure (A) and the design sites (B) of VEGFA-targeted binding proteins

图2 VEGFA结合蛋白的筛选与MB3/VEGFA复合物结构预测示意图Note： A. Yeast surface display of the VEGFA candidate binder library followed by six rounds (sort 0~5) of dual-fluorescence-activated cell sorting. The double-positive cell ratio increased from 0.01% (sort 1) to 32.90% (sort 4) over sequential sorting rounds. B. SEC purification of VEGFA and MB3. C. Determination of the binding affinity between VEGFA and MB3 by BLI. The measured (yellow) and fitted (gray) curves are shown. The 10 μmol/L concentration shown in the legend represents the maximum MB3 concentration in the analyte gradient. D. Binding interface of the MB3/VEGFA complex predicted by AlphaFold3. The side chains of the residues from MB3 (pink) and VEGFA (cyan) that form hydrogen bonds (yellow dashed lines) and a salt bridge (gray dashed line) are also shown.

Fig 2 Experimental screening of VEGFA-binding proteins and schematic diagram of MB3/VEGFA complex structure prediction

图3 MB3对VEGFA介导的HUVECs存活作用的影响Note： A. Effect of different concentrations of VEGFA on HUVECs. B. Effect of different doses of MB3 on VEGFA-treated HUVECs.

Fig 3 Effects of MB3 on VEGFA-induced survival in HUVECs

图4 MB3对VEGFA介导的HUVECs抗凋亡作用的影响Note： A. Western blotting analysis of the expression of BCL-2 and BAX in HUVECs. B/C. Quantification of the ratios of BCL-2/GAPDH (B) and BAX/Bcl-2 (C). D. Detection of the effect of MB3 on VEGFA-treated HUVEC apoptosis by flow cytometry. Q3 (lower right) and Q2 (upper right) quadrants represent early and late apoptotic cells, respectively. E. Quantification of the percentage of apoptotic HUVECs after treatment with VEGFA and MB3.

Fig 4 Effects of MB3 on VEGFA-induced anti-apoptotic effects in HUVECs

图5 MB3对VEGFA介导的HUVECs迁移、体外血管生成及下游磷酸化通路的影响Note： A. Western blotting analysis of the expression of total and phosphorylated forms of AKT and ERK. B. Quantification of the ratios of p-AKT/AKT and p-ERK/ERK. C. Observation of the effect of MB3 on VEGFA-induced HUVEC migration by scratch wound healing assay (×40). D. Quantification of the migration rate. E. Observation of the effect of MB3 on VEGFA-induced HUVEC angiogenesis by tube formation assay (×100). F. Quantification of the total number of junctions, total length, total segment length, and total branching length of VEGFA-induced tube formation in HUVECs after MB3 treatment. 1 px=0.11 μm.

Fig 5 Effects of MB3 on VEGFA-induced migration, angiogenesis, and downstream phosphorylation signaling pathways in HUVECs

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靶向VEGFA结合蛋白的设计与功能表征

Design and functional characterization of VEGFA-targeted binding proteins

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