大鼠GluR2基因启动子控制的萤火虫荧光素酶表达载体的构建

Construction of Firefly Luciferase Expression Vector Driven by Rat GIuR2 gene Promoter

目的构建大鼠GluR2基因启动子控制的萤火虫荧光素酶表达载体，并检测其在神经元中的表达。方法采用PCR方法获得GluR2基因启动子区目的片段（-298～＋283），双酶切后插入到pGL3-Basic载体中构成重组表达载体，使萤火虫荧光素酶报告基因的表达受GluR2启动子控制。将构建的重组表达载体或pGL3-Basic载体分别与内参质粒pRL—CMV（表达海肾荧光素酶）共转染原代培养皮质神经元，24h后用双荧光检测试剂盒测定萤火虫荧光素酶及海肾荧光素酶活性。结果重组表达载体经双酶切及测序鉴定证明构建正确，该重组表达载体在神经元中特异性高表达萤火虫荧光素酶。结论成功构建大鼠GluR2基因启动子控制的萤火虫荧光素酶表达载体，并检测到该载体在神经元中的特异性表达。

Objective To construct a firefly luciferase expression vector driven by rat GluR2 gene promoter and determine its expression in neurons. Methods A 581bp fragment （from ？ 298 to ＋ 283, in which transcription start site is marked as ＋ 1 ） of GluR2 gene promoter was amplified and inserted into pGL3-Basic vector that contains a firefly luciferase reporter gene driven by GluR2 promoter. The recombinant vector or pGL3-Basic vector was co-transfected together with pRL-CMV vector containing renila luciferase reporter gene into primarily cultured cortical neurons. Firefly and renila luciferase activities were analyzed 24h later by use of Dual Luciferase Reporter Assay System. Results Restriction enzyme double digestion and sequencing analysis confirmed correct construction of recombinant expression vector containing GluR2 promoter controlled firefly luciferase reporter gene. High expression of firefly luciferase was detected in neurons. Conclusion A firefly luciferase expression vector driven by rat GluR2 gene promoter is successfully constructed, which specifically expresses firefly luciferase in neurons.