IRX1真核表达载体的构建及在胃癌细胞SGC-7901中的定位

目的 构建pEGFP-IRX1真核表达载体,体外转导至SGC-7901胃癌细胞株,观察IRX1基因转染后蛋白表达与细胞内定位.方法 PCR扩增IRX1全长1443 bp编码序列,将PCR扩增产物连接人pGEM-Teasy载体,经测序确认序列无误后,亚克隆入pEGFP-N1载体,构建pEG-FP-IRX1真核表达载体.采用Lipofectamine 2000转染SGC-7901胃癌细胞株,在荧光显微镜下观察外源性IRX1基因导入后在胃癌细胞内的表达与亚细胞定位.结果 成功构建有绿色荧光蛋白融合的IRX1真核表达载体,外源性IRX1基因在胃癌SGC-7901细胞中表达成功,在荧光显微镜下IRX1表达蛋白定位于细胞核内.结论 pEGFP-IRX1真核表达载体构建成功,并可在细胞内表达,这将为IRX1在胃癌内的功能研究奠定基础.

Construction of IRXI eukaryotic expression vector and its sub-cellular localization in SGC-7901 gastric cancer cell line

Objective To construct the eukaryotic expression vector of pEGFP-IRX1, and observe the IRX1 protein expression and its sub-cellular localization in SGC-7901 gastric cancer cell line. Methods The coding sequence of IRX1 1443 bp was obtained by PCR amplification,then the PCR product was in-serted into the pGEM-Teasy vector. After confirming the DNA sequence, IRX1 amplification product was sub-cloned into pEGFP-N1 vector. Finally, an IRX1 gene eukaryotic expression vector was obtained. The pEGFP-IRX1 expression vector was transfected into SGC-7901 gastric cancer cell line by Lipofectamine 2000,and the expression of pEGFP-IRX1 was detected by GFP fluorescence. Results The recombinant vector was highly expressed in SGC-7901 cells. The green fluorescence of EGFP-1RX1 fusion protein was clearly localized in nuclei of gastric cancer cells. Conclusion pEGFP-IRX1 expression vector was con-structed successfully, and the IRX1 protein was mainly observed in nuclei,which settled a basis for further functional study of gene IRX1 in gastric cancer.