摘要
目的构建并筛选有效的、编码短发夹RNA(shRNA)的增殖诱导配体(APRIL)基因特异性真核表达载体。方法分别设计、合成4对寡核苷酸单链,经退火、连接得到APRIL—shRNA双链,定向克隆至带有H1启动子、绿色荧光蛋白(green fluorescent protein,GFP)基因和Neo基因的pGCsi—H1/Neo/GFP质粒载体中,构建4条含APRIL基因shRNA的pGCsi—H1/Neo/GFP—APRIL—shRNA真核表达载体,转染高表达APRIL的结直肠癌细胞株SW480,G418筛选,荧光显微镜下观察转染效率,FQ—RT—PCR检测APRIL mRNA水平,Western印迹分析APRIL蛋白表达。结果测序证实pGCsi—H1/Neo/GFP—APRIL—shRNA(APRIL shRNA)构建成功,无碱基突变,4种不同shRNA质粒转染细胞后APRIL mRNA弄口蛋白表达差别有统计学显著性意义(P〈0.01)。结论成功地构建APRIL shRNA真核表达载体,敲低效率85%以上,可用于后续的实验研究。
Objective To construct and identify eukaryotic expression vector expressing shRNA sections targeting a proliferation-inducing ligand (APRIL) gene (APRIL-shRNA). Methods Four pairs of oligonucleotides were designed and chemically synthesized. After annealing and ligation, they were directionally inserted into plasmid pGCsi-H1/Neo/GFP with H1 promoter and termination code,the green fluorescence protein (GFP) gene and Neo gene. In this way,4 vectors of pGCsi-H1/Neo/GFP-APRIL-shRNA containing APRIL-shRNA were constructed and they were transfected into the colon carcinoma cell SW480. After screening by G418,the rate of transfection was detected by fluorescence microscope. The inhibition effectiveness of APRIL mRNA and protein was analyzed by FQ-RT-PCR and western blotting respectively. Results The constructed eukaryotic expression vectors effectively suppressed the APRIL expression in transfected cells. The different vectors of 4 short hairpin RNA of APRIL effectively suppressed the expression in SW480 (P〈0.01). Conclusion Constructed and identified a eukaryotic expression vector of short hairpin RNA,specific for APRIL.
出处
《现代检验医学杂志》
CAS
2010年第2期27-30,共4页
Journal of Modern Laboratory Medicine
基金
江苏省卫生厅“科教兴卫”医学重点学科资助项目(编号:XK200723)
南通大学自然科学项目(编号092043).
关键词
增殖诱导配体
短发夹RNA
G418
真核表达载体
a proliferation-inducing ligand
short hairpin RNA
G418
eukaryotic expression vector