目的:构建针对人VEGF基因的小干扰RNA(siRNA)的表达载体,实现CMV启动子调控,转染肿瘤细胞后观察其对VEGF基因的干扰作用,并筛选出有效的VEGF-shRNA.方法:设计三对VEGF靶向的发夹状shRNA,依据设计合成两条互补的寡核苷酸链,退火后连接入质粒pDC311-SV40-RC中,转化扩增后进行序列测定.用脂质体包裹转染人骨肉瘤细胞U-2 OS,分别培养3d和7d后收集细胞培养上清液,ELISA检测上清中VEGF蛋白的表达.结果:将针对VEGF基因的siRNA的双链寡核苷酸片段成功克隆到pDC311-SV40-RC载体,经过阳性菌落PCR鉴定与测序,结果正确;转染U-2 OS细胞后,ELISA方法检测蛋白表达水平证实干扰序列3能有效降低VEGF的表达.结论:成功构建了CMV启动子调控的针对VEGF基因的siRNA载体,转染肿瘤细胞后可抑制VEGF的表达,并筛选出有效的干扰序列.
Objective: To construct VEGF gene-targeted small interfering RNA (siRNA) and its expression vector driven by CMV promoter and to investigate its interference effect. Methods: The VEGF gene-targeted hairpin siRNA was designed,two complementary oligonucleotide strands were synthesized.After annealing,two-strand oligonucleotide was inserted into pDC311-SV40-RC vector,which was then identified by PCR and sequenced.Then human U-2 OS cell line was transfected with the vector using lipofectamine method.Finally,ELISA was performed to evaluate the expression of VEGF protein. Results: PCR-identification of positive clone and sequencing confirmed the vector containing the target siRNA.ELISA showed that compared with the control group,the expression levels of VEGF protein in transfected U-2 OS cells were decreased significantly(P＜0.05). Conclusion: VEGF gene-targeted siRNA and its vector mediated by CMV promoter were successfully constructed,which can reduce the VEGF protein expression after transfecting.
刘晓艳;方红;陈鸿超;蒋筱凌. CMV启动子调控的VEGF-shRNA表达载体的构建及有效干扰序列的筛选[J]. 浙江大学学报（医学版）, 2010, 39(2): 181-186.