Smac-EGFP真核表达质粒的构建

Construction of eukaryotic expression vector for Smac-EGFP

目的构建EGFP融合表达第二线粒体源的半胱氨酸激活物(second mitochondria-derived activator of caspases,Smac)基因的真核表达质粒。方法提取He La细胞总RNA,逆转录合成c DNA,以其为模板,PCR扩增Smac基因,将其插入到p EGFP-N3质粒中,构建重组真核表达质粒p EGFP-N3-Smac,通过脂质体法转染He La细胞,于倒置荧光显微镜下观察转染后质粒在细胞中的分布表达情况,并采用Western blot法鉴定表达的融合蛋白Smac-EGFP。结果成功构建了真核表达质粒p EGFP-N3-Smac,转染后的He La细胞在细胞内膜周围即线粒体部位可见明显、规律的GFP表达;表达的融合蛋白Smac-EGFP相对分子质量约49 000。结论成功构建了重组真核表达质粒p EGFP-N3-Smac,并在He La细胞中表达了融合蛋白,为进一步研究Smac的功能提供了参考。

Objective To construct a eukaryotic expression vector for fusion gene of enhancetl green fluorescent protein （EGFP） and second mitochondria-derived activator of caspases （Stoat）. Methods Total RNA was extracted from HeLa cells and reversely transcribed to eDNA which was used as a template for amplification of Smac gene by RT-PCR. The amplified Smac gene was inserted to vector pEGFP-N3,and the constructed recombinant plasmid pEGFP-N3-Smac was transfected to HeLa cells in mediation of lipofectamine,and observed for distribution and expression under inverted tluorescent microscope. The expressed fusion protein Smac-EGFP was identified by Western blot. Results Euka,Totic expression vector pEGFP-Smac was constructed sucessfully. Obvious and regular expression of GFP was observed in mitochondria of transfected HeLa cells. The relative molecular mass of expressed fusion protein Smac-EGFP was about 49 000. Conclusion Recombinant eukaryotic expression vector pEGFP-N3-Smac was constructed successfully, and fusion protein was expressed in Hel,a cells,which provided a reference for further study on the function of Stoat.