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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。
Objective To clone the gene fragment of human caspase-8 catalytic domain and reconstruct it into two reconstructed human caspase-8 genes with reverse transcripts of large and small subunits. The recombinant plasmids were transfected into HeLa cells and the expression of recombinant human caspase-8 Gene Expression and Its Effect on HeLa Cell Growth. Methods The gene fragment of human caspase-8 catalytic domain was cloned by RT-PCR. The reconstructed human caspase-8 gene with inverted order of large and small subunits was constructed by recombinant PCR. The recombinant plasmid was cloned into the green fluorescent protein (GFP) eukaryotic expression vector pIRES2-EGFP and transfected into HeLa cells. The morphology and structure of the cells were observed by fluorescence microscopy and inverted microscopy. Results The human caspase-8 catalytic domain gene fragment was successfully cloned by RT-PCR and three reconstructed human caspase-8 genes and their eukaryotic expression vectors were constructed. After transfection of HeLa cells, the expression of reconstituted human caspase-8 gene leads to the death of HeLa cells. Conclusion The expression of reconstructed human caspase-8 gene in HeLa cells can effectively induce HeLa cell death.