| 肠出血性大肠埃希菌O157:H7 TCCP蛋白与宿主细胞IRTKS蛋白的相互作用 |
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| 引用本文： | 蒋明明,顾江,王海光,鲁东水,张卫军,罗萍,童文德,邹全明,毛旭虎. 肠出血性大肠埃希菌O157:H7 TCCP蛋白与宿主细胞IRTKS蛋白的相互作用[J]. 免疫学杂志, 2011, 0(2): 149-153 |
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| 作者姓名： | 蒋明明 顾江 王海光 鲁东水 张卫军 罗萍 童文德 邹全明 毛旭虎 |
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| 作者单位： | 第三军医大学医学检验系(药学院)临床微生物及免疫学教研室;解放军第94797部队卫生队; |
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| 基金项目： | 国家自然基金(30670107) |
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| 摘 要： | 目的通过体外结合实验验证EHEC O157:H7内膜素受体偶联细胞骨架蛋白重复片段与人肠上皮细胞IRTKS蛋白SH3结构域之间的相互作用,并制备二者蛋白复合体,为利用晶体学方法研究其相互作用奠定基础。方法将IRTKS蛋白的SH3结构域cDNA序列克隆至pET30a表达质粒,转化BL21(DE3),诱导表达纯化SH3(IRTKS)蛋白;制备TCCP重复片段TC-CP(3R)蛋白;将SH3(IRTKS)与TCCP(3R)按照不同比例混合后,非变性丙烯酰胺凝胶电泳鉴定两者相互结合后产物;利用分子筛层析收集标准蛋白、TCCP(3R)、SH3(IRTKS)和TCCP(3R)-SH3(IRTKS)复合体的出峰体积,回归计算复合物的分子量,分析蛋白相互结合的比例。结果成功克隆表达了SH3(IRTKS)重组质粒;并制备了高纯度的SH3(IRTKS)和TCCP(3R)蛋白;非变性丙烯酰胺凝胶电泳结果证实TCCP(3R)与SH3(IRTKS)发生相互作用形成了蛋白质复合体;复合体的分子量约为33 000,二者相互结合的比例约为1:1。结论本研究为TCCP重复片段与SH3(IRTKS)相互作用提供了直接证据,同时复合体的制备,为利用晶体学研究两者的相互作用奠定了基础。
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| 关 键 词： | EHEC O157:H7 TCCP IRTKS 重组表达 蛋白质复合体 |
Interaction between TCCP of enterohemorrhagic Escherichia coli O157:H7 and IRTKS protein from human in vitro |
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| JIANG Mingming,GU Jiang,WANG Haiguang,LU Dongshui,ZHANG Weijun,LUO Ping,TONG Wende,ZOU Quanming,MAO Xuhu. Interaction between TCCP of enterohemorrhagic Escherichia coli O157:H7 and IRTKS protein from human in vitro[J]. Immunological Journal, 2011, 0(2): 149-153 |
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| Authors: | JIANG Mingming GU Jiang WANG Haiguang LU Dongshui ZHANG Weijun LUO Ping TONG Wende ZOU Quanming MAO Xuhu |
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| Affiliation: | JIANG Mingming,GU Jiang,WANG Haiguang,LU Dongshui,ZHANG Weijun,LUO Ping,TONG Wende,ZOU Quanming,MAO Xuhu Department of Clinical Microbiology and Immunology,College of Medical Laboratory Science,Third Military Medical University,Chongqing 400038,China |
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| Abstract: | The aim of the study is to confirm the interaction between Tir couple cytoskeleton protein(TCCP) of enterohemorrhagic Escherichia coli(EHEC) O157:H7 and insulin receptor tyrosine kinase substrate(IRTKS) protein from human intestinal epithelial cells by in vitro binding assay,and to prepare protein complex for crystallographic studies.IRTKS SH3 domain was amplified from the human cDNA and cloned in the prokaryotic expression plasmid pET30a.Then the recombinant plasmid was transformed to E.coli BL21(DE3) and ... |
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| Keywords: | EHEC O157:H7 TCCP IRTKS Recombinant expression Protein complex |
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