突变的SOD1 cDNA与人MAP/MARK1片段在人胚肾293细胞中的相互作用  

Interaction between mutated SOD1 cDNA and segment of Homo sapiens MAP/MARK1 in human embryonic kidney cell 293

作　　者：陈桂生[1] 史树贵[1] 李露斯[1] 陈康宁[1] 袁顺宗[2] 彭旭[2] 吴军[2] 罗高兴[2] 

机构地区：[1]第三军医大学西南医院神经内科,重庆400038 [2]第三军医大学西南医院全军烧伤研究所,创伤,烧伤与复合伤国家重点实验室,疾病蛋白质组学重庆市市级重点实验室,重庆400038

出　　处：《第三军医大学学报》2007年第24期2327-2329,共3页Journal of Third Military Medical University

基　　金：国家自然科学基金(30300116)~~

摘　　要：目的用酵母双杂交方法显示突变的SOD1cDNA与人MAP/MARK1的片段结合,进一步探讨二者能否在哺乳动物表达系统人胚肾293细胞中发生相互作用,以验证酵母双杂交的结果。方法采用PCR方法及双酶切的方法构建含突变SOD1cDNA片段的真核表达质粒pCMV-Myc和含人MAP/MARK1的片段真核表达质粒pCMV-HA,分别或共同转染人胚肾293细胞,进行免疫共沉淀检测。结果免疫共沉淀结果表明,重组含突变的SOD1cDNA质粒pCMV-Myc分别与空载体pCMV-Myc、pCMV-HA共转染,免疫沉淀物中均未检测到结合蛋白,只有重组含突变SOD1cDNA真核表达质粒pCMV-Myc与重组含人MAP/MARK1的片段真核表达质粒pCMV-HA共转染时,在免疫沉淀物中能检测到结合蛋白,表现在蛋白免疫印迹上有特异性目的条带。结论突变的SOD1cDNA与人MAP/MARK1片段在人胚肾293细胞中存在蛋白水平的相互作用。Objective By using yeast two-hybrid system, an interaction is proven existing between mutated SOD1 cDNA and segment of Homo sapiens MAP/MARK1. In the present study the interaction between the two proteins was further determined in human embryonic kidney cell 293 （ HEK293 ） to verify the results of yeast two-hybrid system. Methods Mammalian expression vectors pCMV-Myc containing mutated SOD1 cDNA and pCMV-HA containing segments of Homo sapiens MAP/MARK1 were constructed by PCR and double enzyme digestion. They were transfected to HEK293 cells and examined by coimmunoprecipitation. Results The coimmunoprecipitation experiments showed when the recombinated plasmid pCMV-Myc with mutated SOD1 cDNA was cotransfected with plasmid pCMV-Myc or plasmid pCMV-HA, there was no binding protein observed in HEK293 cells. However, when the recombinated plasmid pCMV-Myc with mutated SOD1 cDNA and recombinated plasmid pCMV-HA with segment of Homo sapiens MAP/MARK1 were cotransfected into HEK293 cells, the binding protein was detected. Conclusion The mutated SOD1 cDNA interacts with segment of Homo sapiens MAP/MARK1 at protein level in HEK293 cells.

关 键 词：突变SOD1 CDNA 人MAP/MARK1 免疫共沉淀 相互作用 

分 类 号：R322.61[医药卫生—人体解剖和组织胚胎学] R394-33[医药卫生—基础医学]