摘要
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。
Objective:To obtain apoptin gene and to induce tumor cell (HeLa) apoptosis. Methods:Apoptin gene was amplified by PCR from CAV TK5803 genomic DNA,and was then cloned into pCDNA3.1/His/Topo vector. After confirming by DNA sequencing, apoptin gene was subcloned into pCIneo vector.Recombinant plasmid pCDNA3.1/His/Topo apoptin and pCIneo apoptin were transformed into HeLa cells by Lipofectamine reagent. Three days after transfection,the HeLa cell was stained by Honchest 33258 and apoptosis was analysed by fluorescence microscope. Results and Conclusions: The full length apoptin gene was cloned by PCR and inserted into pCDNA3.1/His/Topo vector. Sequence analysis demonstrated that it was same as published apoptin gene sequence. Three days after transfection, HeLa cell turned into apoptosis. [
出处
《军事医学科学院院刊》
CSCD
北大核心
2001年第2期85-87,90,共4页
Bulletin of the Academy of Military Medical Sciences
基金
国家自然科学基金!资助项目 (3 980 0 178)