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目的:观察人上皮细胞生长因子受体2(HER2)靶向重组的活性截短型Bid(tBid)融合蛋白对骨肉瘤SOSP-9607细胞的促凋亡作用。方法:将抗HER2单链抗体基因e23sFv、与绿脓杆菌外毒素(PE)的转膜结构域基因(PEII)和tbid基因连接,构建成immuno-tbid(e23sFv-PEII-tbid)基因,将其克隆入真核表达载体pCMV中,转染SOSP-9607细胞,间接免疫荧光法检测目的蛋白表达和细胞形态学变化,通过AnnexinⅤ染色、流式细胞仪检测观察其促凋亡作用。结果:转染SOSP-9607细胞后,间接免疫荧光染色检测出tB id的表达,SOSP-9607细胞出现明显的固缩,核浓缩等凋亡特征。AnnexinⅤ染色、流式细胞仪检测可见明显的凋亡细胞,凋亡率为42%,对照组细胞仅6%,表明细胞膜表面有磷脂酰丝氨酸外翻,提示重组immuno-tbid基因表达后有促凋亡作用。结论:重组immuno-tbid基因可在转染的SOSP-9607细胞中表达,并诱导其细胞凋亡。
OBJECTIVE: To observe the pro-apoptotic effect of HER2-targeting truncated Bid (tBid) fusion protein on osteosarcoma SOSP-9607 cells. Methods: The anti-HER2 single chain antibody gene e23sFv was ligated with the PEII and tbid genes of Pseudomonas aeruginosa exotoxin (PE) to construct the immuno-tbid (e23sFv-PEII-tbid) Cloned into the eukaryotic expression vector pCMV, transfected into SOSP-9607 cells, the expression of the target protein and cell morphological changes were detected by indirect immunofluorescence. The apoptosis of cells was observed by Annexin Ⅴ staining and flow cytometry. Results: After transfection with SOSP-9607 cells, the expression of tB id was detected by indirect immunofluorescence staining. The apoptotic features of SOSP-9607 cells such as condensation and nuclear condensation were observed. Annexin Ⅴ staining showed that apoptotic cells were detected by flow cytometry. The apoptotic rate was 42% in the control group and only 6% in the control group, indicating that there was phosphatidylserine valgus on the surface of the cell membrane, indicating that there is pro-apoptotic after recombinant immuno-tbid gene expression Dead effect. Conclusion: Recombinant immuno-tbid gene can be expressed in transfected SOSP-9607 cells and induce apoptosis.