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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 a high expression system of neuritin gene and observe the expression of neuritin after transfection of Schwann cells. Methods According to the rat neuritin mRNA sequence, the coding sequence (CDS region) was synthesized in vitro and inserted into the pGH vector to ligate with the GV208-green fluorescent protein (GFP) lentiviral vector fragment. The recombinant clones were identified by restriction enzyme digestion and sequencing . The positive clones were extracted, transfected into 293T cells and packaged into lentivirus to infect rat Schwann cells. Schwann cells were divided into three groups: blank control group (A group), nonsense interference group (B group), high expression group (C group). Real-time quantitative PCR and Western blotting were used to detect the expression of neuritin. Results Rat neuritin was correctly inserted into lentiviral vector. The neuritin mRNA level in group C was significantly higher than that in group A and group B (P <0.01). A fusion protein of neuritin protein and GFP was detected. Conclusion The neuritin lentiviral high expression system was constructed successfully. The expression of neuritin in the transfected Schwann cells increased.