EGFP-线粒体蛋白导入载体的构建及功能验证

目的 构建能够将外源蛋白导入真核细胞线粒体的真核表达载体,并且通过增强型绿色荧光蛋白的表达进行示踪.方法 利用多重PCR扩增法将线粒体导肽序列与EGFP序列融合在一起,并插入真核表达载体pcDNA3.1,构建成真核表达载体pcDNA5.1-EGFP.将P53基因分别插入pcDNA5.1-EGFP和pcDNA3.1构建成pcDNA5.1-P53和pcDNA3.1-P53载体.经酶切和测序验证后在脂质体介导下分别将上述载体转染293T细胞,一方面在荧光显微镜下比较绿色荧光蛋白与细胞色素C在细胞内的分布情况,另一方面通过免疫荧光法比较P53蛋白在细胞内的定位.结果 绿色荧光的表达分布与细胞色素C具有一致性,且转染pcDNA5.1-P53载体的细胞内P53蛋白集中在胞质线粒体中,而转染pcDNA3.1-P53载体的细胞内P53蛋白主要分布在细胞核内.结论 成功构建能够将外源蛋白导入线粒体的真核表达载体.

Construction of the mitochondrial protein imported vector with EGFP and confirmation

Objective To construct a eukaryotic expression vector which can transport foreign protein into mitochondria.Methods The mitochondrial transit peptide’s sequences were fused with EGFP through multiplex PCR amplification and inserted into the eukaryotic expression vector pcDNA3.1,constructing the eukaryotic expression vector pcDNA5.1-EGFP.Then the P53 gene was inserted into pcDNA5.1-EGFP and pcDNA3.1,constructing pcDNA5.1-P53 and pcDNA3.1-P53 vector.After verification by restriction enzyme digestion and sequencing,the plasmids were transfected into 293T cells for profiling the distribution of EFGP and cytochrome C in the cell,and the positioning of P53 protein in the cell.ResultsThe distribution of EGFP was consistent with cytochrome C,and the P53 protein expressed by pcDNA5.1-P53 was concentrated in mitochondria in the cytoplasm,while the vector pcDNA3.1-P53 was mainly distributed in the nucleus.Conclusions The eukaryotic expression vector was successfully constructed,which may transport foreign protein into the mitochondria.