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目的:探讨靶向活性caspase-6融合蛋白对HER2阳性胶质瘤细胞系U251的促凋亡作用。方法:利用白喉毒素furin酶识别序列(Fdt)连接抗HER2单链抗体基因e23sFv和重构型caspase-6(RC6)基因,连接入pCMV载体,构建重组基因的真核表达载体pCMV-e23sFv-Fdt-RC6。脂质体转染HER2阳性胶质瘤U251细胞系,Western blot检测目的蛋白的表达。流式细胞术(FCM)检测转染后U251细胞的凋亡率,MTT法检测目的基因转染后对U251细胞增殖的影响。结果:双酶切及基因测序证实,pCMV-e23sFv-Fdt-RC6载体被成功构建。Western blot检测到转染表达载体48h后的U251细胞中有活性caspase-6的表达。FCM检测到实验组细胞凋亡率比对照组显著增高。MTT实验观察到转染载体pCMV-e23sFv-Fdt-RC6的U251细胞的增殖被明显抑制。结论:e23sFv-Fdt-RC6融合蛋白对HER2阳性胶质瘤细胞系U251有明显的促凋亡作用。
Objective: To investigate the pro-apoptotic effect of targeting active caspase-6 fusion protein on HER2 positive glioma cell line U251. Methods: The anti-HER2 single chain antibody gene e23sFv and the reconstructed caspase-6 (RC6) gene were ligated into the pCMV vector using the diphtheria toxin furin enzyme recognition sequence (Fdt) to construct the eukaryotic expression vector pCMV-e23sFv-Fdt -RC6. The liposome was transfected into HER2 positive glioma U251 cell line, and the expression of the target protein was detected by Western blot. The apoptosis rate of U251 cells was detected by flow cytometry (FCM). The proliferation of U251 cells was detected by MTT assay. Results: Double enzyme digestion and gene sequencing confirmed that pCMV-e23sFv-Fdt-RC6 vector was successfully constructed. The expression of active caspase-6 in U251 cells was detected by Western blot 48 h after transfection. FCM detected apoptosis rate of experimental group than the control group was significantly higher. MTT assay showed that the proliferation of U251 cells transfected with pCMV-e23sFv-Fdt-RC6 was significantly inhibited. Conclusion: The e23sFv-Fdt-RC6 fusion protein has obvious apoptosis-inducing effect on HER2 positive glioma cell line U251.