摘要
目的探讨由野生型人caspase-3大小亚基颠倒构建的重组型caspase-3的促细胞凋亡活性。方法用RT-PCR法获得人caspase-3基因。通过重组PCR进行改造,构建小亚基位于大亚基之前的两种重组型caspase-3基因,它们所对应的蛋白质的N端,分别带有或没有其自身识别的四肽序列。将上述基因克隆入绿色荧光蛋白(GFP)表达载体pIRES2-EGFP中,转染人HeLa细胞,在荧光显微镜和电镜下观察转染细胞的形态和结构。结果成功地获得了野生型caspase-3基因及两种重组型caspase-3基因。构建了重组型caspase-3基因的表达载体。转染HeLa细胞后,重组型caspase-3基因在细胞中得到表达,随后引起细胞荧光强度下降,生长状况变差甚至死亡。电镜观察显示,许多细胞呈现凋亡的典型特征。结论重组型caspase-3可促进HeLa细胞的凋亡。
Aim To investigate the activity of accelerating HeLa cell apoptosis of recombinant caspase 3, constructed from reversal of two subunits of wild type caspase 3. Methods Human caspase 3 gene was obtained by RT PCR, and was transformed into two recombinant caspase 3 genes by settling the small subunit prior to the large ones by recombinant PCR. Two types of recombinant caspase 3 were different because one had N terminal sequence that could be recognized and cleaved by itself, while the other had not. Both genes were cloned into GFP expression vector pIRES2 EGFP and transfected into HeLa cells. The transfected cells were observed under fluorescent and electronic microscopes. Results Wild type and recombinant caspase 3 genes were cloned successfully. HeLa cells transfected by GFP expression vectors of recombinant caspase 3 expressed target proteins, and displayed much weaker fluorescence and poor growth condition and typical features of apoptosis. Conclusion Both recombinant caspase 3 can effectively accelerate HeLa cell apoptosis.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2001年第3期218-221,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家杰出青年科学基金资助
No.39925036
军队杰出青年科学基金资助
No.98J009
