人RASSF2基因的真核表达载体的构建及其表达

The Construction and Expression of Eukaryotic Expression Plasmid for Human RASSF2

目的 构建人RASSF2基因的真核表达载体 ,为研究RASSF2在肿瘤细胞凋亡、细胞周期阻滞等方面的作用奠定基础。方法 采用RT PCR自正常人外周血单个核细胞RNA中扩增出人RASSF2基因的全长cDNA ,利用DNA重组技术将其插入到真核表达载体 pcDNA3.1/HisC中获得重组质粒。用脂质体将重组质粒转染人胰腺癌细胞株PANC 1,RT PCR检测其表达。结果 酶切图谱分析及基因测序证明人RASSF2表达片段已被完整、正确的插入到 pcDNA3.1/HisC中 ,RT PCR结果显示转染细胞RASSF2表达水平上调。结论 成功构建了RASSF2基因的真核表达载体 pcDNA3.

Objective To construct eukaryotic expression plasmid of human Ras association domain family 2 gene for further study on RASSF2 function on tumor cell apoptosis and cell cycle arrest.Methods The RASSF2 gene fragment was amplified by RT PCR (from normal human peripheral blood )and cloned into the eukaryotic expressing vector pcDNA 3.1 /His C by DNA recombination techniques, and then PANC 1 cells were transfected with reconstructive plasmid by liposome mediated gene transfer method. RT PCR was used to detect the expression of RASSF2 gene.Results After the identification of restriction analysis and sequencing, the reconstructive plasmid was confirmed which contained the correct and entire nucleotide sequence of the RASSF2 DNA in pcDNA 3.1 /His C. The expression of RASSF2 in RNA level ascend in PANC 1 cells transfected with reconstructive plasmid.Conclusion The pcDNA3.1 RASSF2 plasmid was constructed and expressed in vitro.