| 杨峰 孙文娟 刘丽丽 杜德伟 孙脊峰△.线粒体蛋白运输能力测定工具建立[J].,2017,17(2):247-251 |
| 线粒体蛋白运输能力测定工具建立 |
| Construction a Tool to Monitor Mitochondrial Import Ability |
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| DOI： |
| 中文关键词: SLC25A6 线粒体 急性肾损伤 |
| 英文关键词: SLC25A6 Mitochondrion Acute kidney injury |
| 基金项目:国家自然科学基金项目（81400690） |
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| 中文摘要: |
| 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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