乙酰肝素酶shRNA双基因共表达真核载体的构建和筛选

Construction and identification of eukaryotic expression vector expressing double shRNA sections targeting heparanase gene

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，将两两随机问隔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 fluoreseent 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．

AIM： To construct and identify eukaryotic expression vector expressing double shRNA sections targeting heparanase gene （Hpa-shRNA）. METHODS： Six pairs of oligonucleotides were designed and chemically synthesized. After randomly connecting an oligonucleotide and another one with 4 -8 bases pairs interval, they were directionally inserted into plasmid pGenesil-1 with respectively U6 promoter and termination code, the common green fluorescence protein （EGFP） gene and Neo gene. In this way, 3 vectors of pGenesil-1-Hpa-shRNA containing 2 sections of Hpa-shRNA were constructed and they were transfected into the ovarian cancer cell SKOV3. The rate of transfection was detected by flow cytometry and fluorescence microscope. The inhibition effectiveness of Hpa protein was analyzed by immunohistochemical staining. RESULTS： The constructed eukaryotic expression vectors effectively suppressed the Hpa expression in transfected cells. The 3 vectors of different short hairpin RNA of Hpa effectively suppressed the expression in SKOV3 （ P 〈 0.01 ）. CONCLUSION： We have constructed a eukaryotic expression vector of shRNA, specific for Hpa and have constructed and identified a eukaryotic expression vector of double-gene short hairpin RNA for Hpa.