利用pGenesil-1.0构建人Dna2真核表达干扰载体

目的:构建人基因Dna2干扰(RNAi)的真核表达载体.方法:根据GenBank上人Dna2的cDNA序列设计并合成两条单链寡核苷酸,退火以后,插入质粒pGenesil-1.0多克隆位点的BamH I和Hind III之间进行重组,构建重组干扰载体,转化DH5α后测序鉴定出阳性菌株.重组质粒以脂质体法转染HeLa细胞,提取Dna2蛋白以免疫印迹法检验干扰效果.结果:设计的目的干扰序列5’-catagccagtagtattcgatg-3’可明显抑制Dna2在HeLa细胞的表达.结论:利用pGenesil-1.0构建的人Dna2干扰载体适用于该基因功能研究.

Construction of Human Dna2 Eukaryotic Expression Vector for RNA Interference by Using pGenesil-1.0

Objective:To construct an eukaryotic expression vector encoding an shRNA targeting human Dna2.Methods:According to the human Dna2cDNA sequence in GenBank,two single oligo nucleotides were designed and synthesised.After annealing,they were inserted into the multiple cloning site of plasmid pGenesil-1.0between BamH I and Hind III to construct the shRNAeukaryotic expression vector.The recombinant plasmid were transformed into DH5αand the positive strains were identified by sequence analysis.The recombinant plasmid were transfected into Hela cells with Lipofectamine 2000,the expression of Dna2protein was detected by western blotting to detect the effect of the RNA interference.Results:The designed interference sequence 5'-catagccagtagtattcgatg-3'could obviously inhibit the expression of Dna2in Hela cells.Conclusion The Human Dna2shRNAeukaryotic expression vector constructed by using pGenesil-1.0was suitable for further studying the function of Dna2.