NSE启动子调控的pNSE-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 a NSE-Promoter Regulatory Eukaryotic Bicistronic Expression Vector PNSE-IRES2-EGFP-p27

目的:构建并鉴定带NSE启动子的Flag-p27和EGFP双顺反子真核表达载体pNSE-IRES2-EGFP-p27,转染原代培养的神经元,观察其表达。方法:通过质粒抽提,电泳、酶切、连接、转化等多种基因工程技术,经多步亚克隆后完成能同时表达p27和EGFP基因的神经元特异性表达载体pNSE-IRES2-EGFP-p27;转染体外培养的神经元观察EG-FP的表达及通过免疫荧光细胞化学技术观察p27的表达。结果:经酶切和测序鉴定,成功构建真核表达载体pNSE-IRES2-EGFP-p27;转染pNSE-IRES2-EGFP-p27后神经元中可见EGFP的表达,且EGFP阳性细胞中p27表达水平明显增高。结论:NSE启动子能启动目的基因p27和EGFP在神经元的表达,连于Flag-p27的下游EGFP可作为报告基因,指示p27的表达情况。带启动子NSE的Flag-p27和EGFP双顺反子真核表达载体的构建为进一步研究神经元细胞周期与神经系统疾病的关系,并为寻找神经系统疾病的有效基因治疗途径奠定基础。

Objective： To construct Flag-p27 and EGFP co-expressed pNSE-IRES2-EGFP-p27 plasmid, and to detect its expression in cultivated neurons. Methods： By way of genetic engineering techniques as plasmid extraction, agarose gel electrophoresis, restriction enzymolysis, connection, transformation of competent cells, the recombinant plasmid of pNSE-IRES2-EGFP-p27 was obtained. The constructed plasmids were transfected to cultivated neurons with liposome and the expression of EGFP and p27 was detected by immunocytochemistry. Results： pNSE-IRES2-EGFP-p27 was successfully constructed and verified by enzymolysis and sequencing. Confocal microscopy revealed that after the plasmids were transfected to neurons, the expression of EGFP could be detected and the expression of p27 was increased significantly in EGFP positive cells. Conclusion：The vectors containing the NSE promoter resulted in the EGFP and p27 expression in neurons. EGFP can be as a report gene to indicate the expression of p27. The construction of pNSE-IRES2-EGFP- p27 lies a foundation for future research on the relationship between cell cycle of neurons and nervous system disease.