一个ALS家系突变SOD1编码蛋白的相互作用蛋白筛选

Search for interesting proteins of mutation SOD1 encoded protein with FALS through the yeast-two-hybrid system

目的:筛选与突变SOD1编码蛋白相互作用的蛋白。方法:应用Clontech酵母双杂交系统3,对人胎脑cD-NA文库进行筛选。把构建成功的质粒pGBKT7-mSOD1cDNA转染酵母菌AH109,再与含有已克隆到pACT2载体上的人胎脑cDNA文库的酵母菌Y187杂交融合。突变的SOD1cDNA与相应的人胎脑cDNA片段编码的蛋白发生相互作用后,可激活基因的表达,筛选出阳性克隆,对阳性克隆的质粒进行测序分析。在GenBank中作匹配及生物信息学分析。结果:共筛选出15个阳性克隆,其中包括9个为已知蛋白,6个为未知蛋白。其中包括PTPN2(protein tyrosinephos phatase,non-receptor type2)。结论:突变SOD1可能通过这些相互作用的蛋白进行相互调控,改变了正常SOD1酶的信号传导通路,从而使机体患病。

Objective:to search for mutation SOD1 interacting proteins.Methods:A DNA fragment encoding the full length mutation SOD1cDNA was inserted into pGBKT7-BD vecter and fused in-frame to the DNA-binding domain of GAL4.It was used a bait to screen pretransformed Human fetal brain MATCHMAKER cDNA library,with the cDNA fragment inserted into pACT2 vecter and fused in-frame to the GAL4 activation domain.If mSOD1 interacted with a protein encoded by a cDNA fragment in the yeast,the transcription of reporter gene coul...