摘要
目的 观察诱导表达的颗粒酶B融合蛋白对HeLa细胞形态和生长的作用。方法 用重组PCR法将活性型颗粒酶B基因序列的 5’端连接绿脓杆菌外毒素 (PE)的部分转位肽编码序列。所获融合蛋白基因克隆入pIND诱导表达载体后 ,与辅助质粒pVgRXR共转染HeLa细胞 ,经G4 18和zeocin筛选建系。间接免疫荧光检测蜕皮激素诱导后目的蛋白的表达 ,并通过电镜、TUNEL染色、细胞计数等方法观察细胞的形态和生长速度的变化。结果 建立了可诱导表达人颗粒酶B融合蛋白基因的HeLa细胞系。蜕皮激素诱导后检测到目的蛋白的表达 ,同时观察到细胞出现多核巨细胞的异常形态 ,细胞生长受到抑制。电镜和TUNEL分析表明 ,颗粒酶B融合蛋白基因的诱导表达使部分细胞呈现凋亡的典型特征。
Objective To investigate apoptosis in HeLa cells induced by expression of human granzyme B fusion protein. Methods Granzyme B fusion protein gene, which consisted of portion of transmembrane sequence of Pseudomonas exotoxin A (PE) and active granzyme B gene, was generated by recombinant PCR and cloned into the inducible expression vector pIND. Then HeLa cells were co-transfected with the resulting expression vector and assistant plasmid pVgRXR, and subjected to G418 and zeocin selection.Expression of target protein on ponasterone A induction was detected by indirect immunofluorescence, and the effect on morphology and growth of HeLa cells was observed by electronic microscopy, TUNEL staining and cell counting. Results The HeLa cells inducibly expressing granzyme B fusion protein were obtained. The target protein was detected in the cytoplasm of the cells after ponasterone A induction, resulting in increased cell size and multinucleation, and growth inhibition was observed. Electronic microscopy and TUNEL analysis showed that the inducible expression of granzyme B fusion protein gene caused typical features of apoptosis. Conclusion Inducible expression of granzyme B fusion protein gene leads to apoptosis of HeLa cells.
出处
《西安交通大学学报（医学版）》
CAS
CSCD
北大核心
2004年第4期329-332,共4页
Journal of Xi’an Jiaotong University（Medical Sciences）
基金
国家高技术"863"计划资助项目 (No .2 0 0 1AA2 1 71 0 1 )
国家杰出青年科学基金 (No .3992 50 36)
全军医药卫生科研基金重点项目 (No.0 1Z0 90 )
