哈萨克族食管癌中cdc42启动子区CpG岛及全基因共构建真核表达载体

Construction of cdc42 gene and promoter CpG island of Hazak′s esophageal cancer in Eukaryotic Expression Vector

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 cdc42 and promoter CpG island of Hazak′s esophageal cancer in Eukaryotic Expression Vector,for further study the effection of methylation of promoter to the gene′ expression and function.Methods cdc42 gene and promoter CpG island with PCR were cloned from Hazak′s esophageal cancer in cDNA and genome DNA expressed positively,after restrictive endonuclease enzymes EcoR I and BamH I cutting,link cdc42 gene with eukaryotic expression vector pEGFP-N1 and transfer into E.coli ER1793.After validated the correctness,cut the recombined vector and the promoter CpG island with enzyme HindⅢ,link the promoter CpG island with recombined vector and transfer into E.coli ER1793.Then transfected the recombined vector into esophageal cancer strain Eca-109.Results DNA sequence which included ORF and promoter CpG island were gained,and successful linked with eukaryotic expression vector pEGFP-N1.By fluorescence microscope we got the green fluorescent protein.Conclusion Eukaryotic expression vector was successfully constructed,it is the basic for further study cdc42 gene expression and function affected by methylation of promoter CpG island.