重构型人caspase-8基因的表达及其对HeLa细胞生长的影响

Expression of recombinant human caspase-8 genes and their effects on growth of HeLa cells

目的 克隆人ｃａｓｐａｓｅ－８催化结构域基因片段，并将其改建成２种大、小亚基基因次序颠倒的重构型人ｃａｓｐａｓｅ－８基因，转染ＨｅＬａ细胞，观察重构型人ｃａｓｐａｓｅ－８基因的表达及其对ＨｅＬａ细胞生长的影响。 方法 用ＲＴ－ＰＣＲ法，克隆人ｃａｓｐａｓｅ－８催化结构域基因片段，经重组ＰＣＲ改造，构建大、小亚基基因次序颠倒的重构型人ｃａｓｐａｓｅ－８基因。将其克隆入绿色荧光蛋白（ＧＦＰ）真核表达载体ｐＩＲＥＳ２－ＥＧＦＰ，转染ＨｅＬａ细胞，用荧光显微镜和倒置显微镜镜观察细胞的形态和结构。 结果用ＲＴ－ＰＣＲ法，成功地克隆了人ｃａｓｐａｓｅ－８催化结构域基因片段，构建了３种重构型人 ｃａｓｐａｓｅ－８基因及其真核表达载体。转染ＨｅＬａ细胞后，重构型人ｃａｓｐａｓｅ－８基因的表达可导致ＨｅＬａ细胞死亡。 结论重构型人ｃａｓｐａｓｅ－８基因在ＨｅＬａ细胞中的表达可以有效地引起ＨｅＬａ细胞死亡。

Aim To clone the catalytic domain gene of human caspase-8 and two kinds of human caspase-8 genes which their large subunits were preceded by their small subunits were reconsctructed . Expression of the reconstructed human caspases-8 genes in HeLa cells and their effects on growth of HeLa cells were evaluated.Methods The catalytic domain gene of human caspase-8 was cloned by RT-PCR, two kinds of reconstructed human caspase-8 genes which their large subunits were proceded by their small subunits were reconstructed by recombinant PCR. The reconstructed human caspases-8 genes were cloned into eukaryotic expression vector pIRES2-EGFP. HeLa cells transfected with reconstructed human caspases-8 were observed under fluorescent and reverse electronic microscopes. Results The catalytic domain gene of human caspase-8 was cloned by RT-PCR, three kinds of reconstructed human caspases-8 genes and their eukaryotic expression vectors were reconstructed successfully. After being transfected, HeLa cell appeared cell death when the reconstructed human caspase-8 genes expressed.ConclusionThe expression of reconstructed human caspases-8 genes can accelerate HeLa cell death efficiently.