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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
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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.
作者 陈桂生 史树贵 李露斯 陈康宁 袁顺宗 彭旭 吴军 罗高兴
机构地区 第三军医大学西南医院神经内科 第三军医大学西南医院全军烧伤研究所
出处 《第三军医大学学报》 CAS CSCD 北大核心 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)~~
关键词 突变SOD1 CDNA 人MAP/MARK1 免疫共沉淀 相互作用 mutated SOD1 cDNA Homo sapiens MAP/MARK1 coimmunoprecipitation interaction
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—医学遗传学]
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参考文献12

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二级参考文献24

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第三军医大学学报
2007年 第24期

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