线粒体蛋白运输能力测定工具建立

Construction a Tool to Monitor Mitochondrial Import Ability

目的:克隆SLC25A6基因入pcDNA3.0表达载体中,同时给基因两端加入HA和Myc标签,为探索SLC25A6基因作为工具研究线粒体生物合成作用在急性肾损伤产生机制提供基础。方法:根据NCBI人SLC25A6 mRNA序列设计引物,通过酶切连接反应将SLC25A6插入到pcDNA3.0中,成功构建pcDNA3.0-1xHA-SLC25A6-1xMy后,经酶切和测序验证正确后,将重组表达质粒转染入人Hela细胞,通过Western-Blot验证重组质粒的表达情况。结果:pcDNA3.0-1xHA-SLC25A6-1xMyc表达质粒测序比对完全正确,转染Hela细胞后可见明显的表达条带。结论:成功构建了N端带有HA tag,C端带有Myc tag的SLC25A6基因表达载体pcDNA3.0-1xHA-SLC25A6-1xMyc,通过转染Hela细胞证明其能正确表达,为研究SLC25A6作为线粒体生物合成能力的监测工具探索线粒体在急性肾损伤中的机制奠定了基础。

Objective： In order to clone human SLC25A6 into vector pcDNA3.0, and add HA tag to the N terminal, Myc tag to the C terminal respectively, lay the foundation for further study of SLC25A6 as a tool to monitor the mitochondrial import ability. Methods：The PCR primers were designed based on the SLC25A6 mRNA sequence deposited in NCBI and then they were digested by double restriction enzymes and inserted into vector pcDNA3.0. After verification by digestion and sequencing, the constructed plasmid pcDNA3.0-1xHA-SLC25A6-1xMy was transfected into human Hela cells. SLC25A6 expression was verified by Western-blot. Results： The cloned recombination plasmid as tool pcDNA3.0-1xHA-SLC25A6-1xMyc was confirmed by enzyme digestion and DNA sequencing. SLC25a6 gene can be successfully expressed in human Hela cells. Conclusion： Human SLC25A6 tagged with HA in N terminal and Myc in C terminal respectively pcDNA3.0-1xHA-SLC25A6-1xMyc were successfully cloned, and it was well expressed in Hela cells, which provides basic tools for the following studies about SLC25A6 as a tool to monitor mitochondrial import ability and study mitochondrial crucial role in acute kidney injury.