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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。
OBJECTIVE: To clone the Nox4 gene into pLenti6.3 lentiviral expression vector and provide experimental basis for exploring the role of Nox4 in ROS production. Methods: According to the NCBI human Nox4 mRNA sequence, primers were designed. Nox4 was inserted into the entry vector pENTR3C by restriction enzyme ligation. After pENTR3C-Nox4 was successfully constructed, Nox4 and EGFP tag were inserted into lentiviral vector pLenti6 by LR reaction. 3, the recombinant plasmid was transfected into Hela cells after digestion and sequencing. The expression of Nox4 was verified by Western-Blot. The localization of Nox4 in cells was verified by immunofluorescence. Results: The sequence of the entry vector and the expression plasmids was completely correct. The Hela cells transfected with HeLa cells showed obvious expression bands and mainly localized in the organelle endoplasmic reticulum. CONCLUSION: The Nox4 gene lentivirus with EGFP tag was successfully constructed and transfected into Hela cells, which can be correctly expressed and localized in the endoplasmic reticulum, which lays the foundation for the study of the role of Nox4 in the regulation of ROS production.