摘要
目的:构建抗HER2重组融合蛋白基因ScFv/tBid,并探讨其对骨肉瘤E10细胞的促调亡作用。方法:通过间接免疫荧光染色、流式细胞仪(FCM)检测E10细胞膜表面HER2的表达。将抗HER2单链抗体基因e23sFv与铜绿假单胞菌外毒素PE的转膜结构域基因(PEⅡ)和tBid基因连接,构建抗HER2重组融合蛋白基因ScFv/tBid,将其克隆入真核表达载体pCMV中构建重组pCMV-ScFv/tBid载体,转染骨肉瘤E10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,Annexin V染色流式细胞术及TUNEL法检测E10细胞的凋亡情况。结果:流式细胞仪检测到E10细胞膜表面有HER2的表达。成功构建重组融合蛋白基因质粒pCMV-ScFv/tBid。重组质粒转染E10细胞后,间接免疫荧光双标记染色检测到E10细胞中tBid的过表达;细胞色素C在细胞质中出现;细胞出现明显的固缩、核浓缩等形态特征。Annexin V染色后流式细胞仪检测可见实验组细胞凋亡率较对照组明显升高(16.1% vs 4.5%);TUNEL染色显示,E10细胞出现典型的凋亡特征。结论:重组抗HER2融合蛋白基因ScFv/tBid可以在转染的骨肉瘤E10细胞中表达,并诱导骨肉瘤细胞发生凋亡。
Objective：To construct a fusion gene ScFv/tBid against HER2 and investigate its pro-popotic effect on osteosarcoma cell line E10. Methods： HER2 expression on the surface of E10 cells was detected by immunofluorescent staining and flow cytometry （FCM）, then e23sFv fragment, a single-chain HER2 antibody, was linked with a PE translocation domain （PE aa253-364） and tBid. The recombinant tBid gene was cloned into a pCMV plasmid to obtain pCMV-ScFv/tBid, which was then transfected into E10 cells. Immunofluorescent staining was used to examine the expression of target protein and morphological changes of cells. Meanwhile, the pro-apoptotic effect of ScFv/tBid gene was analyzed by Annexin V-FITC staining and TUNEL staining. Results： Flow cytometry showed HER2 expression on cell surface, and the recombinant plasmid, pCMV-ScFv/tBid, was successfully constructed and transfected into E10 cells. Overexpression of tBid protein was detected in E10 cells as revealed by immunofluorescent staining; and shrinkage and nuclear condensation were also noticed in E10 cells. Annexin V-FITC staining and FCM revealed that the apoptosis rate of E10 cells was 16.1% after transfection with pCMV-ScFv/tBid; the apoptosis rate in the control cells was 4.5%. TUNEL staining showed typical apoptosis characteristics of E10 cells after transfection. Conclusion： The recombinant anti-HER2 fusion gene, ScFv/tBid, can be expressed in E10 cells transfected with pCMV-ScFv/tBid, and subsequently induce apoptosis.
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
2008年第2期105-109,共5页
Chinese Journal of Cancer Biotherapy
基金
国家杰出青年科学基金资助项目(No39925036)
国家自然科学基金重点项目(No30330610)
国家自然科学基金资助项目(No30471988)
中国博士后基金资助项目(No2005038259)~~
关键词
TBID
人表皮生长因子受体2
融合蛋白
骨肉瘤细胞
细胞凋亡
BH3 interacting death agonist （tBid）
human epidermal growth factor receptor-2（HER2）
fusion protein
osteosarcoma cell
apoptosis
