摘要目的 探讨靶向人类表皮生长因子受体2(Her-2)的曲妥珠单抗(Trastuzumab)与UL16结合蛋白2(ULBP2)的融合蛋白(T-ULBP2)激活自然杀伤(NK)细胞,杀伤乳腺癌细胞SK-BR-3的治疗策略及治疗效果.方法 使用重叠聚合酶链反应(PCR)法通过G4S柔性肽将曲妥珠单抗重链C末端及人ULBP2胞外区基因连接,构建人工程载体pCDNA3.1中,通过脂质体转染法将质粒转入人胚肾细胞HEK-293中进行表达.流式细胞术检测T-ULBP2对乳腺癌SK-BR-3细胞的结合能力.噻唑蓝(MTT)法检测T-ULBP2是否保留亲本曲妥珠单抗对SK-BR-3细胞的增殖抑制能力.通过抗体依赖的细胞介导的细胞毒作用(ADCC)实验验证T-ULBP2激活NK细胞杀伤肿瘤细胞的效果.流式细胞术检测T-ULBP2是否可以诱导NK-92细胞的活化.结果 成功表达融合蛋白T-ULBP2.流式细胞术检测T-ULBP2与乳腺癌细胞SK-BR-3的结合率为79.5％,与亲本曲妥珠单抗结合率(72.9％)相当.结论 成功构建并表达了T-ULBP2.T-ULBP2具有与亲本曲妥珠单抗相似的乳腺癌细胞结合能力和抑制肿瘤增殖能力.

abstractsObjective Evaluating the therapeutic effect of the Trastuzumab fusion antibody T-u-nique long-16 binding protein 2 (ULBP2) on breast carcinoma cell SK-BR-3.Methods A new fusion protein T-ULBP2 gene was obtained by overlapping PCR method containing the heavy chain of patented Trastuzumab,G4S linker and the unique long-16 binding protein 2 (ULBP2).The gene segment of T-ULBP2 was inserted into expression vector pCDNA3.1 and the vector was transected into HEK-293 cell.The binding activity of T-ULBP2 or Trastuzumab to SK-BR-3 was detected by flow cytometry.Methyl thiazol tetrazolium (MTT) assay was used to detect the inhibition of cell growth.Antibody dependent cellular cytotoxicity (ADCC) assay was performed to measure the killing effect of PBMCs or NK-92 on SK-BR-3 tumor cells.The activation of NK-92 cells was tested and verified by flow cytometry.Results The eDNA sequences of Trastuzumab were fused with human ULBP2 to form T-ULBP2 by overlap PCR using a G4S as a linker.T-ULBP2 was purified and followed by Western blotting analysis.T-ULBP2 showed high binding activity on SK-BR-3 cells and the binding rate was 79.5％,similar to that of Trastuzumab (72.9％).Conclusion Comparing with the parent antibody Trastuzumab,the fusion protein T-ULBP2 showed a similar binding activity and anti-tumor activity in vitro on SK-BR-3.