人肾脏相关基因Nox4过表达慢病毒载体的构建与定位检测

目的: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Nox4 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-Nox4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Nox4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。

Construction and Localization of Lentiviral Vectors Targeting for Human kidney related Nox4 Gene

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.