人上皮细胞黏附分子shRNA表达载体的构建及鉴定

Construction of ShRNA Eukaryotic Recombinant Plasmid Targeting Human EpCAM

目的以人上皮细胞黏附分子(epithelial cell adhesion molecule,Ep CAM)基因为靶基因,构建shRNA重组表达载体。方法根据Gen Bank中Ep CAM序列(BC014785.1)的mRNA设计4组shRNA序列,插入p SGU6/GFP/Neo载体,构建重组载体,转化至Top10感受态细胞,经卡那霉素筛选并挑取阳性克隆,进行PCR鉴定及DNA测序分析。结果 PCR鉴定及DNA测序结果显示重组载体Ep CAM-p SGU6/GFP/Neo-shRNA构建成功。结论成功构建了干扰人Ep CAM基因的重组表达载体,为研究Ep CAM分子的生物学功能打下基础。

Objective To construct the recombinant eukaryotic vector expressing short hairpin RNA（shRNA） section targeting hu- man EpCAM gene. Methods According to the sequence of human EpCAM mRNA （GenBank ID ： BC014785.1 ） , four groups of shRNA sequence were designed and cloned into pSGU6/GFP/Neo vector to construct the recombinant plasmids, which were transformed into Topl0 competent cells, screened by kanamycin. The positive clones were selected, and the extracted plasmids were identifed by PCR analysis and gene sequencing. Results PCR analysis and DNA sequencing results showed that the eukayotic expression vector of EpCAM - pSGU6/GFP/Neo - shRNA were successfully constructed. Conclusion The eukaryotic expression vector expressing shRNA targetin human EpCAM is successfully constructed, which provide a foundation for further study on the function of EpCAM.