靶向表皮生长因子受体shRNA质粒表达载体的构建和鉴定

目的构建靶向表皮生长因子受体（EGFR）的短发卡状RNA（shRNA）质粒表达载体并进行鉴定。方法设计两个小分子干扰RNA（siRNA）序列，体外合成DNA模板引物，与Pgenesil-1质粒构建成编码shRNA的表达载体，进行酶切鉴定和测序，再转染人大肠癌LoVo细胞，进行荧光摄像和G418抗性筛选。结果构建的质粒表达载体完全符合设计要求，转染成功的细胞在荧光显微镜下显示为绿色，G418可以筛选出阳性克隆。结论成功构建了编码EGFR—shRNA的质粒表达载体，并可以进行稳定筛选。

Establish and identify plasmid expressive vector coding shRNA targeting epidermal growth factor receptor

Objective] To establish and identify the plasmid expressive vector coding short hairpin RNA (shRNA) targeting epidermal growth factor receptor (EGFR) of human gene.Methods] Two sequences of small interference RNA (siRNA) were designed,DNA template-primer of EGFR gene was synthesized in vitro and the plasmid expressive vectors coding shRNA were established with Pgenesil-1 plasmid,then were identified by digestion and tested for the sequence. The human colon cancer LoVo cells were transfected with plasmid vector,and then were taken fluorescence photographs and selected by G418. Results] The digestion identification of plasmid vectors confirmed that DNA template-primer was successfully inserted in the plasmid and the sequence was in conformity with the designed result.After the plasmid vector with green fluorescent protein was transfected to the cells,it was seen as a green fluorescent wave. The positive clones were also successfully selected.Conclusion] Plasmid expressive vectors coding EGFR-shRNA are successfully established and capable of stable transfection.