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(P<0.01)。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。
Objective To construct lentiviral neuritin over-expression system and identify it in the transfected rat Schwann cells （SCs）in vitro. Methods According to the mRNA sequence of rat neuritin gene, the coding sequence （CDS） of neuritin gene was synthesized, constructed into the pGH vector,and then linked with the GV208-GFP lentiviral vector. The recombinants were identified by restriction digestion and DNA sequencing. The plasmids of positive clones were transfected into 293T cells,and then infected into rat SCs. The SCs were divided into three groups of A（normal control group） ,B（nonsense vector control group） and C（neuritin over-expression group）. The expressions of neuritin of SCs were assayed by RT-qPCR and Western blot. Results Compared with groups of A and B, group C showed that the expression of neuritin gene was significantly up-regulated at mRNA levels （P〈0. 01） and the neuritin protein and GFP fusion protein were detected using Western blot. Conclusion Lentiviral neuritin over-expression system has been successfully constructed and transfected into SCs with significantly up-regulated neuritin expression.
出处
《江苏医药》
CAS
北大核心
2013年第9期993-995,共3页
Jiangsu Medical Journal
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)
