波形蛋白干扰质粒shRNA Vim的构建及鉴定

Construction Authentication of shRNA Vimentin Recombinant Plasmid

目的以波形蛋白基因为靶基因,构建波形蛋白干扰质粒shRNA Vim并验证其干扰效果。方法根据GenBank中大鼠波形蛋白的mRNA序列设计shRNA序列,插入至pSilencer 4.1载体,构建重组质粒,转化到Top10感受态细胞,经氨苄青霉素筛选挑取阳性克隆并进行测序分析。结果DNA测序分析显示干扰质粒shRNA Vim构建成功,免疫印迹法显示该干扰质粒能有效地抑制波形蛋白的表达(P<0.001)。结论成功构建波形蛋白干扰质粒shRNA Vim,为研究波形蛋白的生物学功能打下基础。

Objective To construct the shRNA vimentin recombinant plasmid through taking vimentin gene as the target gene. Methods The shRNA sequence was designed according to the sequence of rat vimentin mRNA from GenBank,then was cloned into pSilencer4.1 vector to construct the recombinant plasmid,which was transformed into Top10 competent cells and screened by ampicillin.The positive clones were selected and identified by DNA sequencing analysis.Results The DNA sequencing analysis showed that the shRNA vimentin recombinant plasmid was constructed successfully.Western blot analysis showed that shRNA Vim recombinant plasmid had a noteworthy suppression effect on vimentin expression(P<0.001).Conclusion The shRNA Vim recombinant plasmid is constructed successfully,which can provide a foundation for further study on the biological function of vimentin.