| 杨峰 孙文娟 李占亭 杜德伟 孙脊峰.人肾脏相关基因Nox4过表达慢病毒载体的构建与定位检测[J].,2016,16(2):230-234 |
| 人肾脏相关基因Nox4过表达慢病毒载体的构建与定位检测 |
| Construction and Localization of Lentiviral Vectors Targeting for Humankidney related Nox4 Gene |
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| DOI： |
| 中文关键词: Nox4 慢病毒载体；ROS |
| 英文关键词: Nox4 Lentiviral vector ROS |
| 基金项目:国家自然科学基金项目（81400690） |
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| 中文摘要: |
| 克隆Nox4基因入pLenti6.3 慢病毒表达载体，为探索Nox4基因在ROS 产生中的作用提供实验基础。方法：根据
NCBI人mRNA 序列设计引物，再利用酶切连接反应将Nox4插入到入门载体pENTR3C中，成功构建pENTR3C- 后，
通过LR 反应，将Nox4和EGFP tag 插入到慢病毒表达载体pLenti6.3 中，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 验证的表达情况，免疫荧光验证Nox4在细胞内的定位情况。结果：入门载体及表达质粒测序
比对完全正确，转染Hela 细胞后可见明显的表达条带，并且主要定位于细胞器内质网中。结论：成功构建了带有EGFP tag 的
Nox4基因慢病毒重组表达载体，转染Hela 细胞后，其能正确表达并定位于内质网中，为研究Nox4在调节ROS 产生中的作用奠
定了基础。 |
| 英文摘要: |
| Objective:To clone human Nox4 into a lentiviral over-expression vector pLenti6.3, lay the foundation for further study
of Nox4 functions in ROS production.Methods:Based on the Nox4 mRNA sequence deposited in NCBI. Primers were designed, and
then they were digested by double restriction enzymes and inserted into the Entry clone pENTR3C Nox4 plasmid. After successfully
constructing pENTR3C-Nox4 plasmid, the and EGFP tag were inserted into pLenti6.3 expression vector by LR reactions. After
verification by digestion and sequencing, the constructed plasmids was transfected into human Hela cells. expression were verified
by Western-blot and cellular localization wes verified by IF based on the EGFP tag signal.Results:The cloned recombination
plasmid were confirmed by enzyme digestion and DNA sequencing. Nox4 gene could express in human Hela cells, besides, Nox4 mainly
localized in endoplasmic reticulum (ER).Conclusion:The lentiviral vector over-expressed human Nox4 tagged with EGFP were
successfully cloned, and it was well expressed in Hela cells, besides, it was mainly localized in the ER, which provided basic tools for the following studies about Nox4 roles for ROS production. |
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