论文部分内容阅读
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。
OBJECTIVE: To construct a recombinant expression vector targeting Cyr61 targeting siRNA, and to lay the foundation for the study on the inhibition of Cyr61 gene expression on lung injury induced by mechanical ventilation. Methods: According to the nucleotide sequence of Cyr61 gene provided by GenBank database, three DNA sequences capable of transcribing siRNA were designed and named as Cyr61-1 siRNA, Cyr61-2 siRNA and Cyr61-3 siRNA. At the same time, a nonspecific sequence was designed as negative Control. According to the design and synthesis of their respective oligonucleotide strands, after annealing connected to pGenesil1.1 vector, after transformation and amplification sequence determination. Four recombinant plasmids were transfected into lung cancer A549 cells. The expression of Cyr61 was detected by RT-PCR and Western blot respectively at the mRNA and protein levels. Results: The recombinant plasmid expressing Cyr61 targeting siRNA was confirmed by restriction enzyme analysis and sequencing. The inhibitory rates of Cyr61 mRNA and protein expression were 60.54% and 52.97%, respectively. Conclusion: Cyr61 target siRNA recombinant expression vector was constructed successfully and could significantly inhibit the expression of Cyr61 gene.