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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。

英文摘要:

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.

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