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目的:探讨人表皮生长因子受体-2(human epidermal growth factor receptor 2，HER2)蛋白的哪种结构域更适合激发赫赛汀抗体依赖的细胞介导的细胞毒性(antibody-dependent cell-mediated cytotoxicity，ADCC)效应。方法:真核表达五种包含HER2胞外段不同结构域的蛋白，即HER2-Fc、HER2-His、T23-561-Fc、T276-T652-Fc、T276-T652-His。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis，SDS-PAGE)对蛋白相对分子质量及纯度进行检测。酶联免疫吸附实验(enzyme linked immunosorbent assay，ELISA)检测这些蛋白与赫赛汀的结合能力。自然杀伤(natural killer，NK)细胞活化实验比较这五种蛋白增强赫塞汀ADCC效应的能力差异。结果:成功获得纯度较高的蛋白。ELISA结果显示HER2-Fc，HER2-His和T276-T652-His都表现出较好的结合能力，T276-T652-Fc结合较差，T23-561-Fc则完全不结合。此外，HER2-Fc、HER2-His、T276-T652-Fc、T276-T652-His、T23-561-Fc的EC50值分别为0.16、0.17、0.16、0.15、1.12。NK细胞活化实验结果显示HER2-Fc和T276-T652-His能显著增强赫赛汀刺激NK细胞脱颗粒和分泌干扰素-γ(interferon-γ，IFN-γ)的效应，HER2-Fc组CD107a表达量最高可达61.50%，IFN-γ的分泌可达38.10%。T276-T652-His组CD107a的表达最高可达63.00%，IFN-γ的分泌可达43.10%，与HER2-Fc作用相当。该效应呈浓度梯度依赖性，蛋白低浓度0.5 μg/mL时，赫赛汀的ADCC效应降低，且组间差异有统计学意义(n t值分别为4.21、4.24、6.51、3.17、3.44、3.05、3.34、2.82、8.55、11.52、4.50、2.96、5.58、6.03、3.77、4.42，n P值均<0.05)。n 结论:验证了T276-T652-His具有增强赫赛汀ADCC功能的作用，且与HER2-Fc相当，为后续将HER2蛋白用于肿瘤免疫治疗研究打下了基础。“,”Objective:To exlore which domain of human epidermal growthfactor receptor 2(HER2) protein is more suitable to stimulate the antibody-dependent cell-mediated cytotoxicity (ADCC) effect of Herceptin.Methods:Eukaryotes express five proteins containing different domains of HER2 extracellular segment, namely HER2-Fc, HER2-His, T23-561-Fc, T276-T652-Fc and T276-T652-His. The molecular weight and purity of the protein were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The binding of these proteins to Herceptin was detected by enzyme linked immunosorbent assay (ELISA). Natural killer (NK) cell activation assay was used to compare the ability of these five proteins to enhance the ADCC effect of Herceptin.Results:High purity protein was successfully obtained. ELISA results showed that HER2-Fc, HER2-His and T276-T652-His all had good binding ability, T276-T652-Fc binding was poor, and T23-561-Fc did not bind at all. The EC50 of HER2-FC, HER2-His, T276-T652-Fc, T276-T652-His and T23-561-Fc are 0.16, 0.17, 0.16, 0.15 and 1.12. NK cell activation assays showed that HER2-Fc and T276-T652-His significantly enhanced Herceptin-stimulated NK cell degranulation and interferon-γ(IFN-γ) secretion in a concentration gradient-dependent manner. At low protein concentration(0.5 μg/mL), the ADCC effect of Herceptin decreased significantly, and the difference between groups was statistically significant (n t were 4.21, 4.24, 6.51, 3.17, 3.44, 3.05, 3.34, 2.82, 8.55, 11.52, 4.50, 2.96, 5.58, 6.03, 3.77 and 4.42, respectively, all n P values <0.05). The expression of CD107a in the HER2-Fc group was up to 61.50%, and the secretion of IFN-γ was up to 38.10%. The expression of CD107a in the T276-T652-His group was up to 63.00%, and the secretion of IFN-γ was up to 43.10%, which was comparable to HER2-Fc.n Conclusion:It was validated that T276-T652-His had the ability to enhance the ADCC function of Herceptin and is comparable to HER2-Fc, laying the foundation for the subsequent use of HER2 protein in tumor immunotherapy studies.