Cyr61基因siRNA表达载体的构建及沉默作用研究

Construction of Cyr61 si RNA expression vector and its Characteristic investigation

目的:构建Cyr61靶向siRNA重组表达载体,为探讨抑制Cyr61基因表达对机械通气所致肺损伤的研究奠定基础。方法:根据GenBank数据库提供的Cyr61基因核苷酸序列,选择设计3条能转录siRNA的DNA序列,命名Cyr61-1 siRNA,Cyr61-2 siRNA,Cyr61-3 siRNA,同时设计1条非特异性序列作为阴性对照。据此设计合成各自的寡核苷酸链,退火后连接入pGenesil1.1载体,转化扩增后进行序列测定。4种重组表达载体转染肺癌A549细胞,逆转录RT-PCR和Western blot法分别在mRNA和蛋白水平检测Cyr61的表达。结果:经酶切鉴定和测序结果证实Cyr61靶向siRNA重组表达载体构建成功,它对大肠癌细胞Cyr61 mRNA和蛋白的表达抑制率分别为60.54%和52.97%。结论:Cyr61靶向siRNA重组表达载体构建成功并能显著抑制Cyr61基因的表达。

AIM：To construct si RNA recombinant expression vector targeting Cyr61 gene,and to screen the stably transfected cell clone.METHODS： Three si RNAs sequences based on the sequence of Cyr61mRNA in the GenBank were designed and synthesized,and one scrambled siRNA sequences served as negative control.The cDNA was synthesized and inserted into plasmid pGenesil1.1.The plasmids was sequenced to confirm the inserted sequence.RT-PCR and Western blot analysis was used to assess the levels of Cyr61 mRNA and proteins after the constructed plasmids have been transfected into A549 cells.RESULTS： It was confirmed by restriction endonuclease and sequence analysis that siRNA recombinant expression vector targeting Cyr61 gene was constructed successfully.Inhibition ratio of Cyr61 siRNA at mRNA and protein levels were 59.2% and 52.97%.CONCLUSION： The siRNA recombinant expression vector targeting Cyr61 gene has been constructed successfully and can inhibite the expression of Cyr61 gene in A549 cells significantly.