NF-κB启动子调控的pNF-κB-IRES2-EGFP-p27双顺反子真核表达载体的构建及表达分析

Construction and Identification of NF-κB-promoter Regulatory Eukaryotic Bicistronic Expression Vector pNF-κB-IRES2-EGFP-p27

目的构建并鉴定携带核转录因子κB(nuclear factor-κB,NF-κB)特异性启动子的Flag-p27和增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)双顺反子真核表达载体pNF-κB-IRES2-EGFP-p27,转染经H2O2干预的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs),观察其表达。方法采用基因工程技术,经过多步亚克隆后完成能同时表达p27和EGFP基因的NF-κB特异性启动子双转基因真核表达载体。转染体外培养的经H2O2干预的HUVEs,观察EGFP的表达,并通过免疫荧光细胞化学技术观察p27基因的表达。结果经酶切鉴定和测序鉴定,成功地构建真核表达载体pNF-κB-IRES2-EGFP-p27,转染经H2O2干预的HUVEs后,可见部分细胞有EG-FP的表达,并且在EGFP阳性的细胞中p27基因的表达水平明显增高,而未经H2O2处理的对照组转染pNF-κB-IRES2-EGFP-p27后无EGFP的表达。结论NF-κB启动子能够特异性地激活p27基因的表达。EGFP基因可以指示p27基因的表达情况。由NF-κB特异性启动子启动的p27和EGFP双顺反子真核表达载体的构建为进一步研究细胞周期调控与慢性移植物失功(chronic graft dysfunction,CGD)之间的关系,并为进一步寻找慢性移植物失功的基因治疗途径奠定基础。

Objective To construct Flag p27 and EGFP co-expressed pNF κB-IRES2 EGFP-p27 plasmid, and to detect its expression in cultivated human umbilical vein endothelial cells （HUVECs） pretreated with He O2. Methods By using genetic engineering techniques as plasmid extraction, agarose gel electrophoresis, restriction enzymolysis, connection, transformation of competent cells, the recombinant plasmid of pNF-κB-IRES2-EGFP-p27 was constructed, and transfected into cultivated HU VECs with liposome. The expression of EGFP and p27 was detected by immunocytochemistry. Results pNF-κB-IRES2-EG FP-p27 was successfully constructed and verified by enzymolysis and sequencing. Confocal microscopy revealed that after the plasmids were transfected into HUVECs, the expression of EGFP could be detected, and the expression of p27 was increased significantly in EGFP positive cells. Conclusion The vectors containing the NF-κB promoter resulted in the EGFP and p27 expression in HUVECs. EGFP can be used as a report gcne to indicate the expression of p27. The construction of pNF-κB- IRES2-EGFP-p27 provides a foundation for future research on the relationship between HUVECs proliferation and chronic graft dysfunction.