凋亡蛋白抑制因子livin基因siRNA表达载体构建及转染.docVIP

凋亡蛋白抑制因子livin基因siRNA表达载体构建及转染 　【摘要】 目的：构建3个针对凋亡蛋白抑制因子livin基因mRNA的小干扰RNA ( siRNA)表达载体，然后转染HCT-8/V细胞。为研究livin基因沉默对HCT-8/V细胞耐长春新碱特性的影响奠定基础。方法：合成3对靶基因livin的siRNA寡核苷酸序列通过退火生成转录模板，与线性pGCsi-H1/Neo/GFP质粒连接，转化DH5α钙化菌抽提重组质粒，测序鉴定。用脂质体2000将重组质粒转染HCT-8/V细胞并用G418筛选。结果：转化大肠杆菌涂布平板长出阳性菌落，测序结果与所设计、合成的livin 的siRNA转录模板序列一致；在荧光显微镜下观察到HCT-8/V细胞表达绿色荧光蛋白(GFP)证实重组质粒已转染入细胞并用G418剔除了未转染质粒的细胞。结论：成功构建livin基因的siRNA表达载体，转染并筛选出转染了livin siRNA表达载体的HCT-8/V细胞。 【关键词】 小干扰核糖核酸 质粒 凋亡蛋白抑制因子 转染 [Abstract] Objective: To construct 3 eukaryotic expression vectors of short hairpin RNA (shRNA) against livin gene and then transfect into HCT-8/V cells, and to pave the way to research that gene-silencing of livin in HCT-8/V on trait of vincristine resistance. Methods: Three pairs of oligonucleotide based on livin mRNA sequences were synthesized and annealed to produce transcription templates, and then linked with linear plasmid pGCsi-H1/Neo/GFP to generate siRNA eukaryotic expression vectors. Plasmids were extracted following DH5α strains were transformed. Then insert sequences were identified and recombinants were transfected HCT-8/V cells. Results: The insert sequences were identical with designed shRNA template sequences according to sequencing. Expression of green fluorescent protein in transfected HCT-8/V was observed by fluorescence microscopy. Conclusions: The recombinant vectors were established and transfected into HCT-8/V cells successfully, HCT-8/V cell with expression vectors of livin siRNA was screened by G418 and proliferated. [Key words] Small interfering ribonudeoacid; Plasmid; Inhibitor of apoptosis protein; Transfection 凋亡蛋白抑制因子livin是凋亡抑制蛋白家族成员之一，livin的过表达与肿瘤的发生、发展和肿瘤耐药[1]有关。RNA干扰(RNA interference, RNAi)是一种高效的基因表达阻断技术, 它通过体外合成或体内表达在靶细胞内产生与目的基因同源的含21~23个核苷酸的双链小分子干扰RNA 片段(small interfering RNAs, siRNA )。siRNA 制备方法有多种，若在哺乳动物细胞内持久表达siRNA以期长久抑制靶基因，通常采用表达载体的方法。本研究拟构建3个livin基因siRNA 表达载体,用此载体转染耐长春新碱的结肠癌细胞株HCT-8/V,为研究RNAi技术抑制livin在耐长春新碱结肠癌细胞株HCT-8/V中的表达，进一步探索livin的siRNA对逆转HCT-8/V细胞耐长春新碱的效果从而为研究肿瘤耐药的基因治疗