A-Raf-S214C突变体的构建、表达及其对MAPK信号通路的影响

发布时间：2018-02-11 23:09

本文关键词： A-Raf激酶 MAPK信号通路信号传导　出处：《生物技术通报》2017年12期 　论文类型：期刊论文

【摘要】：为了观察A-Raf的突变体A-Raf-S214C对MAPK(mitogen-activated protein kinase,丝裂原活化蛋白激酶)信号通路的影响,并探讨其诱发肺癌和朗格汉斯增生症的可能分子机制。通过定点突变构建pc DNA5-FRT-TO-A-Raf-S214C突变质粒,并利用该质粒构建稳定表达A-Raf-S214C突变体的HEK293细胞系,之后通过检测下游ERK磷酸化程度,确定该突变体对MAPK信号通路的影响。结果显示,通过实验成功构建了稳定表达A-Raf-S214C突变体的细胞系,在不同浓度的多西环素诱导下,其表达量有所不同。与野生型相比,A-Raf-S214C突变体可明显增强MAPK信号通路中下游信号分子ERK的磷酸化。结果表明A-Raf-S214C突变体是A-Raf激酶的激活突变体,可以导致MAPK信号通路下游信号分子ERK磷酸化明显增强。
[Abstract]:To investigate the effect of A-Raf mutant A-Raf-S214C on the signal pathway of MAPK(mitogen-activated protein kinase, mitogen-activated protein kinase (MAPK), and to explore the possible molecular mechanism of its induction of lung cancer and Langerhans hyperplasia, the mutant plasmid PC DNA5-FRT-TO-A-Raf-S214C was constructed by site-directed mutation. The HEK293 cell line expressing A-Raf-S214C mutant stably was constructed by using the plasmid. The effect of the mutant on MAPK signaling pathway was determined by detecting the phosphorylation of downstream ERK. The cell lines stably expressing A-Raf-S214C mutant were successfully constructed and induced by different concentrations of doxycycline. Compared with the wild type, the A-Raf-S214C mutants can significantly enhance the phosphorylation of the downstream signaling molecule ERK in the MAPK signaling pathway. The results indicate that the A-Raf-S214C mutant is the activated mutants of A-Raf kinase. ERK phosphorylation of downstream signal molecules in MAPK signaling pathway was significantly increased.
【作者单位】：昆明理工大学生命科学与技术学院云南省高校靶点药物筛选与利用重点实验室;
【基金】：国家自然科学基金项目(81460253,U1302225) 云南省人才培养项目(KK23201426014)
【分类号】：R597;R734.2

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