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目的 将人Caspase 6基因大小亚基顺序颠倒 ,表达为重构型Caspase 6分子 ,探讨其对细胞凋亡的促进作用。方法 RT PCR获取人Caspase 6基因 ,测序判断正确后 ,通过重组PCR的方法构建大小亚基顺序颠倒的重构型Cas pase 6 (RevCasp6 )基因。将其克隆入含绿色荧光蛋白 (GFP)基因的真核表达载体 pIRES2 EGFP中 ,转染人宫颈癌细胞系HeLa ,在荧光显微镜下观察细胞形态的变化 ,用电子显微镜进一步观察细胞的凋亡特征。结果 获得了人Caspase 6基因 ,并成功地构建了大小亚基顺序颠倒的RevCasp6基因。以构建的RevCasp6真核表达载体 ,转染HeLa细胞后 ,荧光显微镜观察到细胞生长不良、细胞核结构破坏和细胞死亡。电子显微镜观察显示 ,转染了RevCasp6基因的细胞呈凋亡的典型特征。结论 RevCasp6基因具有诱导HeLa细胞凋亡的作用
Objective To reverse the subunit size of human Caspase 6 gene and express it as a reconstructed Caspase 6 molecule, and to explore its role in promoting apoptosis. Methods The human Caspase 6 gene was obtained by RT PCR. After sequencing, the reconstructed Caspase 6 (RevCasp6) gene was constructed by recombinant PCR. The recombinant plasmids were cloned into the eukaryotic expression vector pIRES2 EGFP containing the green fluorescent protein (GFP) gene and transfected into HeLa cells. The morphological changes of the cells were observed under a fluorescence microscope. The apoptosis characteristics of the cells were further observed by electron microscopy . As a result, the human Caspase 6 gene was obtained and the RevCasp6 gene whose order of size was reversed was successfully constructed. The constructed RevCasp6 eukaryotic expression vector was transfected into HeLa cells, and the cell growth defects, the destruction of the nuclear structure and the cell death were observed under a fluorescence microscope. Electron microscopy showed that the cells transfected with the RevCasp6 gene showed typical characteristics of apoptosis. Conclusion The RevCasp6 gene has the effect of inducing HeLa cell apoptosis