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目的:构建人FOXQ1(homo sapiens forkhead box Q1)基因真核表达载体pAcGFP1-N1-FOXQ1,并在大肠癌细胞系Colo-320中瞬时表达.方法:利用PCR方法从YR Gene装载了FOXQ1基因全长cDNA序列(NM_033260)的质粒中扩增出FOXQ1的cDNA片段,与真核表达载体pAcGFP1-N1连接,重组质粒经PCR、双酶切和测序鉴定无误后,采用Lipofectamine 2000瞬时转染Colo-320细胞,应用荧光显微镜观察转染效率、Western blot检测FOXQ1蛋白表达水平.结果:成功构建了真核表达载体pAcGFP1-N1-FOXQ1,PCR、双酶切和测序鉴定结果均正确,载体能在Colo-320细胞中正确表达FOXQ1蛋白.结论:成功构建FOXQ1真核表达载体,为进一步开展体内、体外实验,研究FOXQ1基因在肿瘤发生发展中的功能奠定了实验基础.
OBJECTIVE: To construct the eukaryotic expression vector pAcGFP1-N1-FOXQ1 of human FOXQ1 gene and to express it in the colorectal cancer cell line Colo-320.Methods: The full-length FOXQ1 gene The cDNA fragment of FOXQ1 was amplified from the cDNA sequence (NM_033260) and ligated with the eukaryotic expression vector pAcGFP1-N1. The recombinant plasmid was identified by PCR, double enzyme digestion and sequencing. The recombinant plasmid was transiently transfected into Colo-320 cells with Lipofectamine 2000 The expression of FOXQ1 protein was detected by Western blot.Results: The eukaryotic expression vector pAcGFP1-N1-FOXQ1 was successfully constructed, and the results of PCR, double enzyme digestion and sequencing were correct, and the vector could be expressed in Colo-320 The FOXQ1 protein was correctly expressed in the cells.Conclusion: The FOXQ1 eukaryotic expression vector was constructed successfully, which laid the foundation of further study on the function of FOXQ1 in tumorigenesis in vivo and in vitro.