目的 了解间日疟原虫VIR14蛋白与细胞间黏附分子-1（ICAM-1）受体的结合情况并分析其关键位点。方法 将重组质粒pET28a-VIR14转化至大肠埃希菌BL21（DE3），经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹（Western blotting）分析间日疟原虫重组VIR14蛋白的表达情况与纯度。通过表面等离子共振（SPR）技术验证VIR14蛋白或多肽与ICAM-1受体的结合情况。预测VIR14以及ICAM-1（28~480 aa）蛋白的结构，综合预测复合物建模分数（pTM）及局部距离差异测试分数（pLDDT），通过cluspro 2.0对接分析得分最高的VIR14和ICAM-1模型之间的相互作用。使用PDBePISA系统和pymol 3.0软件处理对接结果，分析对接位点。通过全球不同流行区数据分析结合位点氨基酸多态性。结合表位竞争抑制实验分析受体与配体相互作用的关键位点。结果 SDS-PAGE结果显示，成功表达并纯化相对分子质量（M_r）约为58 000的VIR14蛋白。SPR实验表明，不同浓度的VIR14蛋白与ICAM-1受体结合响应值呈浓度依赖上升，亲和力值为15.16 μmol/L。Alphafold2结构预测结果显示，VIR14蛋白最佳模型pLDDT和pTM评分分别为78.2和0.73；ICAM-1受体最佳模型pLDDT和pTM值分别为93.8和0.65。VIR14与ICAM-1蛋白之间形成极性键的区域集中在蛋白尾端（212~406 aa）。在含有预测结合位点的2条多肽中，仅P2多肽与ICAM-1结合，亲和力为48.99 μmol/L。竞争抑制实验表明，P2多肽能抑制VIR14蛋白与ICAM-1结合。将P2中的4个关键位点（ASP-310、ARG-314、LYS-316、ASP-318）突变为丙氨酸以改变它们的侧链后，P2丧失了抑制能力。P2多肽在全球338个地理株中高度保守。结论 间日疟原虫VIR14蛋白能与ICAM-1受体紧密结合，4个关键氨基酸位点发挥了重要作用，且在全球种群中具有高度保守性。

Objective To investigate the binding of Plasmodium vivax VIR14 protein to intercellular cell adhesion molecule-1 (ICAM-1) receptor and identify key binding sites. Methods The recombinant plasmid pET28a-VIR14 was transformed into Escherichia coli BL21 (DE3). The expression levels and purity of VIR14 protein were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Binding of VIR14 protein or peptides to ICAM-1 was verified using surface plasmon resonance (SPR). Structures of VIR14 and ICAM-1 (28-480 aa) were predicted, and complex modeling scores (pTM and pLDDT) were integrated. Docking analysis was performed using ClusPro 2.0 to evaluate the interactions between top-scoring VIR14 and ICAM-1 models. Docking sites were analyzed using PDBePISA and Pymol 3.0. Amino acid polymorphisms at binding sites were examined using global isolate data. Key interaction sites were analyzed via epitope competition inhibition assays. Results SDS-PAGE confirmed successful expression and purification of VIR14 protein, with a relactive molecular mass (M_r) of 58 000. SPR showed concentration-dependent binding of VIR14 to ICAM-1, with an affinity of 15.16 μmol/L. According to structural predictions from Alphafold2, the optimal model of the VIR14 protein had a pLDDT score of 78.2 and a pTM score of 0.73. In contrast, the optimal model of the ICAM-1 receptor showed a pLDDT score of 93.8 and a pTM score of 0.65. The regions where polar bonds formed between the VIR14 and ICAM-1 proteins were predominantly located in the C-terminal region of the protein, specifically between the 212 and 406 amino acids. Among the two peptides containing the predicted binding sites, only peptide P2 bound to ICAM-1, with an affinity of 48.99 μmol/L. Competitive inhibition experiments indicated that P2 could inhibit the binding of VIR14 protein to ICAM-1. After replacing the side chains of the four key residues (ASP-310, ARG-314, LYS-316, and ASP-318) with alanine, P2 lost its inhibitory function. P2 was highly conserved across 338 P. vivax global isolates. Conclusion The VIR14 protein of P. vivax exhibits tight binding to the ICAM-1 receptor, with four amino acid residues playing essential roles in this interaction. These key sites demonstrate high conservation across P. vivax global isolates.