pEGFP-N1-APOE ε2重组质粒的构建与鉴定

Construction and verification of pEGFP-N1-APOE ε2 recombinant plasmid

目的构建pEGFP-N1-APOEε2真核表达重组质粒。方法应用基因定点突变手段,根据载脂蛋白E(apolipoprotein E,ApoE)基因cDNA克隆基因序列和表达载体增强绿色荧光蛋白真核表达载体(pEGFP-N1)质粒上的多克隆位点设计引物,对质粒APOE基因cDNA克隆(pMD18-T-APOE3)进行基因定点突变,得到含有APOE2基因的目的片段,将此目的片段插入载体启动子下游,与增强型绿色荧光蛋白(EGFP)的基因融合。重组质粒经菌液PCR、限制性内切酶酶切和测序,鉴定目的基因是否成功插入pEGFP-N1质粒。结果对所得pEGFP-N1-APOEε2质粒插入部分测序结果显示,pEGFP-N1-APOEε2质粒的方向及序列正确,阅读框正确无误,表明APOEε2基因定点突变正确,重组pEGFP-N1-APOEε2质粒构建成功。结论成功构建了pEGFP-N1-APOEε2真核表达重组质粒。

Objective To construct enhanced green fluorescent protein（EGFP）labled eukaryotic expression recombinant plasmid of APOEε2gene.Methods According to the sequence of cDNA cloning gene of APOE gene and the multiple clone site of the expression vector pEGFP-N1 plasmid,two specific pairs of primers were desinged and synthesized by site-directed mutation.PCR amplification was carried out on the pMD18-TAPOE3,and an target fragment was acquired.The target fragment was inserted into the carrier promoter downstream and was fused into EGFP gene fusion.The recombinant plasmid was verified by PCR of the bacterium liquid,restriction enzyme digestion and gene sequencing.Results The inserted part of pEGFP-N1-APOEε2plasmid showed that the direction and sequence pEGFP-N1-APOEε2plasmid and the reading frame were correct.The results showed that site-directed mutagenesis of APOE ε2 was correct and recombinant pEGFP-N1-ApoEε2 plasmid was successful.Conclusions The pEGFP-N1-APOEε2eukaryotic expression recombinant plasmid was successfully constructed