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Construction and identification of eukaryotic expression vector expressing double shRNA sections targeting heparanase gene
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- Author:
- No author available
- Journal Title:
- JOURNAL OF THE FOURTH MILITARY MEDICAL UNIVERSITY
- Issue:
- 1
- DOI:
- 10.3321/j.issn:1000-2790.2006.01.005
- Key Word:
- 乙酰肝素酶;U6启动子;短发夹RNA;真核表达载体
Abstract： 目的:构建并筛选有效的、编码2条shRNA的乙酰肝素酶(Hpa)特异性基因真核表达载体. 方法:分别设计、化学合成6对寡核苷酸单链,经退火、连接和磷酸化后得到Hpa-shRNA双链,将两两随机间隔4～8个碱基后定向克隆入带有各自的U6启动子和终止码、共同的绿色荧光蛋白(green fluorescent protein, EGFP)基因和Neo基因的pGenesil-1中,构建3 条含两段Hpa基因shRNA的pGenesil-1-Hpa-shRNA真核表达载体,转染卵巢癌细胞株SKOV3,流式细胞仪和荧光显微镜下观察转染效率,免疫组化分析Hpa蛋白表达. 结果:酶切与测序证实pGenesil-1-HPSE-shRNA构建成功,无任何碱基突变,3组质粒转染细胞均有Hpa表达的变化,3种不同shRNA质粒转染细胞后Hpa蛋白表达有明显差别. 结论: 构建并筛选了针对Hpa特异性的双基因shRNA真核表达载体pGenesil-1-Hpa-shRNA.
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