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目的构建cdc42基因启动子区CpG岛及全基因共构建真核表达载体,研究cdc42基因启动子区的甲基化状态对该基因表达及功能的影响。方法采用PCR方法从新疆哈萨克族食管癌的cDNA表达阳性的标本中扩增出cdc42基因,以阳性组织基因组DNA扩增出cdc42基因启动子区CpG岛,cdc42基因经双酶切后与真核表达载体pEGFP-N1相连,并转入甲基化酶缺陷的大肠杆菌E.coliER1793中扩增。经测序成功后,将重组质粒及cdc42基因启动子区CpG岛用HindⅢ酶切,连接后再次转入E.coliER1793中扩增。将重组质粒转染至食管癌细胞株Eca-109中,荧光显微镜观察结果。结果 DNA测序证明获得了包含ORF全长的cdc42基因及其启动子区CpG岛,并将双序列成功克隆入真核表达载体pEGFP-N1中。转染后荧光显微镜下观察到绿色荧光蛋白。结论成功构建真核表达载体,为进一步探讨cdc42基因启动子区CpG岛甲基化状态对该基因在哈萨克族食管癌细胞株中的表达及功能的影响奠定了基础。
Objective To construct eukaryotic expression vector of CpG island and all genes of promoter region of cdc42 gene and study the influence of methylation status of cdc42 gene promoter on its expression and function. Methods The cDNA of cdc42 gene was amplified by PCR from the positive expression of Kazakh Kazakh esophageal cancer cDNA. The positive cloned genomic DNA was used to amplify the CpG island of cdc42 promoter. Vector pEGFP-N1 was ligated and transformed into methylase-deficient E. coli E. coli 1793 for amplification. After successful sequencing, the recombinant plasmid and CpG island of the promoter region of cdc42 gene were digested with HindIII, then ligated and amplified again into E. coliER1793. The recombinant plasmid was transfected into esophageal cancer cell line Eca-109, and the results were observed under a fluorescence microscope. Results DNA sequencing proved that the CDC42 gene containing full length of ORF and its promoter region CpG island were obtained. The double sequence was successfully cloned into eukaryotic expression vector pEGFP-N1. After transfection, green fluorescent protein was observed under a fluorescence microscope. Conclusion The eukaryotic expression vector was successfully constructed, which laid the foundation for further study on the methylation status of CpG island in promoter region of cdc42 gene and its function in Kazakh esophageal cancer cell line.