IQ结构域的GTP酶激活蛋白1基因短发卡RNA重组质粒的构建及表达

Construction and expression of IQ-domain GTPase-activating protein 1-short hairpin RNA plasmid

目的构建特异性针对人IQ结构域的GTP酶激活蛋白1(IQ-domain GTPase-activating protein 1,IQGAP1)基因短发卡RNA(short hairpin RNA,shRNA)的重组质粒,检测其在人食管癌KYSE150细胞中的表达。方法以人IQGAP1 mRNA序列为靶向设计合成含有shRNA序列的寡核苷酸,克隆至真核表达载体GV102中,构建重组质粒IQGAP1-shRNA,转化DH5α感受态细胞,挑取阳性克隆测序鉴定。用脂质体2000介导IQGAP1-shRNA转染KYSE150细胞作为IQGAP1-shRNA干扰组,同时设转染载体GV102为对照组,RT-PCR及Western blot法检测IQGAP1基因的干扰效果。结果经测序鉴定,重组质粒IQGAP1-shRNA构建正确。IQGAP1-shRNA干扰组及对照组转染KYSE150细胞均可见绿色荧光蛋白(green fluorescent protein,GFP),两组转染效率分别为(76±5. 780)%和(83±3. 851)%。IQGAP1-shRNA干扰组IQGAP1 mRNA转录水平和蛋白表达水平与对照组比较差异均有统计学意义(P <0. 001),表达抑制率分别为(82±2. 424)%及(77±2. 791)%。结论成功构建了IQGAP1-shRNA真核表达质粒,能特异性降低食管癌KYSE150细胞中IQGAP1基因的表达,为进一步研究IQGAP1基因在食管癌中的功能及靶向治疗作用奠定了基础。

Objective To construct an expression vector for the short hairpin RNA(shRNA)targeting human IQ-domain GTPase-activating protein 1(IQGAP1)gene and observe its expression in KYSE150 cells.Methods The oligonucleotides with shRNA sequences targeting human IQGAP1 mRNA were designed and synthesized and cloned into eukaryotic vector GV102.The constructed recombinant plasmid IQGAP1-shRNA was transformed to competent E.coli DH5α,and the positive clones were screened and identified by sequencing.KYSE150 cells were transfected with IQGAP1-shRNA in mediation of lipofectamine 2000,and determined for the expression level of IQGAP1 by RT-PCR and Western blot,using those transfected with empty vector GV102 as control.Results Recombinant plasmid IQGAP1-shRNA was constructed correctly as proved by sequencing.Green fluorescent proteins(GFPs)were observed in both KYSE150 cells transfected with IQGAP1-shRNA and with empty vector GV102,with transfection efficiencies of(76±5.780)%and(83±3.851)%respectively.Both the mRNA transcription and protein expression levels of IQGAP1 in KYSE150 cells transfected with IQGAP1-shRNA were significantly lower than those with empty vector GV102(P<0.001),while the inhibitory rates were(82±2.424)%and(77±2.791)%respectively.Conclusion Eukaryotic expression vector IQGAP1-shRNA was successfully constructed,which specially inhibited the expression of IQGAP1 gene in KYSE150 cells.It laid a foundation of further study on the role of IQGAP1 gene in esophageal cancer and target therapy of the tumor.