大鼠CREB结合蛋白RNA干扰重组慢病毒载体的构建与鉴定

目的构建大鼠CREB结合蛋白(CREB binding protein,CBP)基因RNA干扰(RNAi)慢病毒载体,并观察该载体对大鼠血管平滑肌细胞(vascular smooth muscle cells,VSMCs)CBP表达的沉默效应。方法设计合成4条CBP基因的siRNA序列,将筛选确定的CBP RNAi有效靶序列与pFU-GW-iRNA载体连接产生CBP shRNA慢病毒表达载体,转化、PCR筛选阳性克隆及测序鉴定。脂质体2000将CBP shRNA慢病毒载体和慢病毒包装质粒共转染293T细胞,完成慢病毒颗粒包装及滴度测定。包装产生的重组慢病毒感染大鼠VSMCs,实时定量RT-PCR检测CBP mRNA的表达,并与未转染及转染阴性对照shRNA慢病毒的细胞进行比较。结果 PCR扩增和测序结果证实,成功构建大鼠CBP shRNA慢病毒载体。经包装产生的慢病毒滴度为2×10~9 TU/ml,与未转染慢病毒及转染阴性对照shRNA慢病毒的细胞比较,CBP shRNA慢病毒转染可使VSMCs的CBP mRNA分别下降88.5%和92.7%。结论成功构建CBP基因RNAi慢病毒表达载体,对VSMCs的CBP表达具有良好沉默作用,为后期基因治疗血管增殖性疾病奠定基础。

Construction and identification of a recombinant lentiviral vector encoding shRNA targeting rat CBP gene

Objective To construct a recombinant lentiviral vector encoding shRNA targeting rat CREB binding protein(CBP) gene,and observe the effects of CBP gene silencing in rat vascular smooth muscle cells (VSMCs).Methods Four shRNA sequences targeting CBP gene were designed and synthesized,and the best one was cloned into the pFU-GW-iRNA vector to construct the lentiviral expression vector containing CBP shRNA(LV-CBP shRNA).LV-CBP shRNA confirmed by PCR and DNA sequencing was subsequently cotransfected with packaging plasmids into 293T cells by Lipofectamine 2000.The titer of virus was detected according to the expression of enhanced green fluorescent protein(EGFP).CBP mRNA expression in VSMCs with or without lentivirus transiection was measured by real-time PCR.Results PCR analysis and DNA sequencing demonstrated that the LV-CBP shRNA was constructed successfully.The titer of concentrated lentivirus was 2×10~9TU/ml.Compared with non-transfectd VSMCs and LV-NC shRNA,the level of CBP mRNA in LV-CBP shRNA decreased by 88.5%,92.7%,respectively.Conclusion The recombinant lentiviral vector encoding shRNA targeting CBP was successfully constructed. LV-CBP shRNA has obvious effect on CBP gene silencing in VSMCs,which provides the basis for future gene therapy of vascular proliferative disease.