基质金属蛋白酶抑制因子-1小干扰RNA慢病毒表达载体的构建与鉴定

目的:构建基质金属蛋白酶抑制因子-1(TIMP-1)小干扰RNA慢病毒表达载体并转染肝星形细胞T6观测其转染效率及对TIMP-1mRNA的沉默效应。方法:用PCR扩增在线确定的TIMP-1特异性小干扰RNA序列,与T载体连接,用BamHI和XhoI分别双酶切并将其粘末端亚克隆入pRNAT-U6.2,用pR-NAT-U6.2的插入鉴定引物进行PCR鉴定并测序;包装产生TIMP-1pRNAT-U6.2/lentisiRNA的慢病毒表达载体,将其转染T6细胞,荧光照相及FQ-PCR检测转染效果及TIMP-1mRNA表达。结果:TIMP-1pRNAT-U6.2/LentisiRNA慢病毒表达载体可显著抑制T6TIMP-1mRNA表达。结论:成功构建能高效转染真核细胞的TIMP-1pRNAT-U6.2/LentisiRNA病毒表达载体,为进一步研究其在体抑制TIMP-1mRNA表达提供了实验工具。

Construction and identification of lentivirus expression vector of small interfering RNA specific for TIMP-1

Objective:To construct lentivirus expression vector of small interfering RNA specific for TIMP-1and evaluate their silencing effect on TIMP-1 mRNA expression in hepatic stellate cell(T6).Methods:The TIMP-1 siRNA gene was amplified with PCR and integrated to PGEM-T vector,cut by double enzyme of BamHI and Xhol,sub-cloned to pRNA-U6.2,identified by inserted expression vector that generated primer of pRNA-U6.2 and undergoing DNA sequencing.Package lentivirus expression vector that generated TIMP-1 pRNA-U6.2/lenti siRNA and transfect it into T6 cell.Fluorescent expression of quantitative TIMP-1 siRNA was observed in T6.TIMP-1 mRNA expression was detected using full quantitative RT-PCR.Results:TIMP-1 siRNA was successfully constructed and the effect of inhibition to TIMP-1 mRNA of T6 was significant.Conclusion:Construction of the lentivirus expression vector that efficiently transfected TIMP-1 pRNA-U6.2/lenti siRNA in eukaryotic cells provides experimental tool for further using it to suppress TIMP-1 Mrna in vitro.