关键词: 表达载体, 绿色荧光蛋白, 线粒体导肽

Abstract: Objective To construct the eukaryotic expression vector which could transport foreign protein into the mitochondria. Its expression could be traced by EGFP(enhanced green fluorescent protein). 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 were inserted into pcDNA5.1-EGFP and pcDNA3.1, constructing pcDNA5.1-p53 and pcDNA3.1-p53 vector. After verification by restriction analysis and sequencing, transfected the plasmids into 293T cells respectively. On one hand, to compare the distribution of EFGP and cytochrome C in the cell under the fluorescence microscopy , and on the other hand to compare the positioning of p53 protein in the cell. Results The 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 is mainly distributed in the nucleus. Conclusion The eukaryotic expression vector was successfully constructed ,which could transport foreign protein into the mitochondria.

Key words: expression vector , green fluorescent protein, mitochondrial transit peptide