摘要
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.
AIM：To construct a eukaryotic expression vector expressing FOXQ1（pAcGFP1-N1-FOXQ1） and investigate its transient expression in colorectal cancer cell line Colo-320.METHODS：The full-length FOXQ1 cDNA sequence was amplified from the plasmid containing full-length FOXQ1 cDNA（NM_033260） and inserted into eukaryotic expression vector pAcGFP1-N1.The resulting pAcGFP1-N1FOXQ1 plasmid was identified by PCR,double digestion and DNA sequencing.The constructed plasmid was then transfected into colorectal cancer cell line Colo-320 using Lipofectamine 2000,and transfection efficiency was assessed by fluorescence microscopy.The expression level of FOXQ1 protein was detected by Western blot.RESULTS：Eukaryotic expression vector pAcGFP1-N1-FOXQ1 was constructed successfully and verified by PCR,double digestion and DNA sequencing.The constructed vector could express mature FOXQ1 protein correctly in colorectal cancer cell line Colo-320.CONCLUSION：Successful construction of the eukaryotic expression vector pAcGFP1-N1-FOXQ1 provides an experimental basis for further study of functions of the FOXQ1 gene in tumorigenesis.
出处
《世界华人消化杂志》
北大核心
2013年第20期1966-1971,共6页
World Chinese Journal of Digestology
基金
国家自然科学基金资助项目
No.81260323
云南省科技厅-昆明医科大学联合基金资助项目
No.2012FB095
云南省教育厅科学研究基金项目
No.2012J004
云南省卫生厅基金资助项目
No.2010NS014~~
