Neuritin基因高表达系统构建及转染大鼠雪旺细胞的鉴定

发布时间：2018-04-17 09:14

本文选题：Neuritin基因 + 慢病毒高表达系统　；参考：《江苏医药》2013年09期

【摘要】：目的构建neuritin基因的高表达系统,观察其转染雪旺细胞后neuritin的表达。方法根据大鼠neuritin mRNA序列体外合成编码序列(CDS区),构建到pGH载体,与酶切后的GV208-绿色荧光蛋白(GFP)慢病毒载体片段进行连接、转化、酶切及测序鉴定重组克隆。提取阳性克隆,转染入293T细胞,包装成慢病毒,以此感染大鼠雪旺细胞。将雪旺细胞分为三组:空白对照组(A组)、无意义干扰组(B组)、高表达组(C组)。实时荧光定量PCR和蛋白免疫印迹实验检测细胞neuritin的表达。结果大鼠neuritin正确插入慢病毒载体,C组neuritin mRNA水平显著高于A组、B组(P0.01)。检测到neuritin蛋白与GFP的融合蛋白。结论成功构建neuritin慢病毒高表达系统;其转染的雪旺细胞neuritin表达升高。
[Abstract]:Objective to construct a high expression system of neuritin gene and observe the expression of neuritin in Schwann cells.Methods according to the rat neuritin mRNA sequence, the pGH vector was constructed and ligated with the GV208-green fluorescent protein (GFP) lentivirus vector. The recombinant clones were identified by restriction endonuclease digestion and sequencing.Positive clones were extracted, transfected into 293T cells and packaged into lentivirus to infect Schwann cells in rats.Schwann cells were divided into three groups: blank control group (group A), meaningless interference group (group B) and high expression group (group C).The expression of neuritin was detected by real-time fluorescence quantitative PCR and Western blot assay.Results the level of neuritin mRNA in group C was significantly higher than that in group A (P 0.01).The fusion protein of neuritin and GFP was detected.Conclusion the high expression system of neuritin lentivirus was successfully constructed, and the neuritin expression in Schwann cells transfected with lentivirus was increased.
【作者单位】：南京医科大学第一附属医院内分泌病科;
【基金】：国家自然科学基金(81070655) 江苏省科技厅基础研究计划(自然科学基金)(BK2009441)
【分类号】：R363

【相似文献】