NOX4报告基因重组质粒的构建及表达调控初探

目的：构建NOx4荧光素酶报告基因质粒载体，探讨NOX4基因表达调控机制。方法：以细胞基因组DNA为模板作，采用PCR扩增NOX4基因上游调控序列（533bp），插入质粒DGL3-basic载体，构建重组质粒pGL3-NOX4，重组质粒经酶切和测序鉴定。将pGL3-NOX4转染A549细胞，通过细胞因子刺激观察报告基因表达水平。结果：酶切及测序证实NOX4报告基因质粒构建成功。转染报告基因的A549细胞以细胞因子刺激，结果显示NOx4表达增强。结论：成功构建了NOX4荧光素酶报告基因质粒载体，为进一步研究NOX4基因的表达规律及生理功能奠定了基础。

Construction and expression analysis of recombinant luciferase reporter plasmid with NOX4 promoter

Objective： To investigate the expression and regulation of NOX4 gene in epithelial cells. Methods： 533 bp of the human NOX4 promoter was amplied with PCR. The amplication product was subcloned into the promoterless pGL3-basic rey luciferase vec- tor to generate reporter plasmid pGL3-NOX4. The reporter plasmid was transfected into A549 cells and cells were stimulated with TNF-α and IFN-γ. Firey luciferase activity from the pGL3-NOX4 reporter vector was measured by the Dual Luciferase assay system. Results： The expression of pGL3-NOX4 reporter vector in A549 cells was up-regulated significantly by proinflammation cytokines TNF-α and/FN-γ. Conclusions.. This report demonstrates that proinflammation cytokines may stimulate NOX4 in epithelium and NOX4 may contribute to the mucosal inflammatory and defensive reaction.