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目的 :克隆凋亡素 (apoptin)的全长基因 ,在肿瘤细胞HeLa中表达并诱导其凋亡。方法 :从CAV病毒基因组TK580 3中通过PCR技术扩增凋亡素基因 ,直接克隆至真核表达载体pCDNA3.1/His/Topo ,序列测定证实其正确性 ,并克隆至pCIneo真核表达载体构建重组表达载体。采用脂质体转染HeLa细胞 ,转染 3d后Honchest332 58染色并于荧光显微镜下观察细胞状态。结果与讨论 :核酸序列分析证实所克隆的凋亡素基因与文献报道的一致 ,将其成功克隆至真核表达载体pCDNA3.1/His/Topo和pCIneo ,构建了真核重组质粒pCIneo apoptin和pCDNA3.1/His/Topo apoptin ,转染HeLa细胞 3d后可见明显的细胞凋亡 ,而只转染空质粒的对照组则较少。表明凋亡素能够有效地诱导HeLa细胞凋亡。本研究为进一步研究凋亡素诱导肿瘤细胞凋亡的分子机制打下了基础
Objective: To clone the full-length gene of apoptin and express it in HeLa cells and induce apoptosis. METHODS: The apoptin gene was amplified by PCR from the genomic DNA of TK580 3 and cloned into the eukaryotic expression vector pCDNA3.1/His/Topo. The sequence was confirmed by the sequence analysis and cloned into pCIneo eukaryotic expression vector. Recombinant expression vectors. HeLa cells were transfected with liposome, and after 3 days transfection, the cells were stained with Honchest33258 and observed under fluorescence microscope. Results and discussion: Nucleic acid sequence analysis confirmed that the cloned apoptin gene was identical to that reported in the literature. It was successfully cloned into eukaryotic expression vectors pCDNA3.1/His/Topo and pCIneo to construct eukaryotic recombinant plasmids pCIneo apoptin and pCDNA3. In .1/His/Topo apoptin, obvious apoptosis was observed after transfection of HeLa cells for 3 days, while the control group with only transfected empty plasmid was less. It was shown that apoptin can effectively induce HeLa cell apoptosis. This study lays the foundation for the further study of the molecular mechanism of apoptosis induced by apoptin in tumor cells.