Runx1-shRNA重组质粒的构建与表达

Construction and Expression of Runx1-shRNA Expressiong Plasmid

本研究目的在于构建特异性下调人Runx1基因的shRNA表达质粒。设计并合成人Runx1基因的siRNA,通过DNA重组技术将目的序列插入到pSuper质粒,采用菌落PCR、限制性内切酶酶切技术及测序方法对其进行筛选与鉴定。转染该质粒于人肺腺癌细胞A549,通过实时荧光定量PCR技术和Western blot方法分别从基因及蛋白水平对其进行功能鉴定。实验结果表明我们所构建的pSuper-Runx1-shRNA质粒能够有效抑制A549细胞Runx1mRNA和蛋白的表达,抑制率分别为33%和50%。

This study aims to construct the plasmid of human Runxl and observe its possible effects on RunxI gene expression in human pulmonary adenocarcinoma cells （A549）. The shRNA sequence targeting human Runxl was designed and synthesized, then inserted into pSuper plasmid by DNA recombination technology. The recombinant plasmid was confirmed by bacterial colonies PCR, enzyme digestion analysis and DNA sequencing. A549 cells were transfected by Runxl shRNA plasmid. The inhibition efficiency of pSuper-Runxl-shRNA plasmid on Runxl at mRNA level and protein level were measured with real-time PCR and Western blot. The results of real-time PCR and Western blot indicated that the mRNA and protein levels of Runxl in A549 cells were inhibited by the pSuper-Runxl- shRNA expression plasmid, and the inhibition rate were a3% and 50%, respectively.