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1443bp编码序列,将PCR扩增产物连接入pGEM-Teasy载体,经测序确认序列无误后,亚克隆入pEGFP—N1载体,构建pEGFP-IRX1真核表达载体。采用Lipofectamine2000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。
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 inserted 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-IRX1 fusion protein was clearly localized in nuclei of gastric cancer cells. Conclusion pEGFP-IRX1 expression vector was constructed successfully,and the IRX1 protein was mainly observed in nuclei,which settled a basis for further functional study of gene IRX1 in gastric cancer.