肠出血性大肠杆菌效应因子z2151的E3泛素连接酶活性鉴定  

The E3 ubiquitin ligase activity identification of enterohemorrhagic Escherichia coli z2151 effector

作　　者：陈芳红[1,2] 李涛[2] 李崭[2] 孙超尘 王慧[1,2] 

机构地区：[1]安徽医科大学军事医学科学院微生物流行病研究所,合肥230032 [2]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《安徽医科大学学报》2016年第8期1077-1080,共4页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81401643)

摘　　要：目的利用基因工程方法原核表达z2151蛋白,并鉴定其E3泛素连接酶活性。方法以肠出血性大肠杆菌O157∶H7为模板,PCR扩增目的基因,构建重组表达载体p ET22b-z2151,转化BL21(DE3),诱导蛋白表达,采用镍柱亲和纯化获取目的蛋白;通过体外泛素化实验检测蛋白泛素连接酶活性。结果亲和纯化得到较高浓度和纯度的z2151蛋白,其在体外泛素化反应中能够促使泛素小分子形成多聚泛素链,即有E3泛素连接酶活性;而且对E2泛素结合酶具有一定的选择特异性。结论原核表达质粒p ET22b-z2151构建成功,目的蛋白z2151具有泛素连接酶的体外泛素化能力,并且针对不同的E2酶亲和力不同,这些都为后续功能机制研究奠定了基础。Objective To express z2151 protein in E. coli by genetic engineering methods, and to identify its E3 ubiquitin ligase activity. Methods The full-length z2151 were cloned from EHEC O157 ： H7 by PCR and were in- stated into plasmid pET22b to construct the recombinant expression vector pET22b-z2151. The recombinant plasmid was transformed into E. coli BL21 （DE3） strains which were incubated for expression. And the recombinant protein was purified using Ni2 ＋ NTA Sephrose; the in vitro ubiquitination method was used to identify its ubiquitin ligase activity. Results The z2151 protein of high concentration and purity was obtained, and found its E3 ubiquitin ]ig- ase activity by which it could help ubiquitin to form polyubiquitin chains in vitro activity; what＇ s more,the z2151 protein selectively interacted with human E2 ubiquitin conjugating enzymes. Conclusion The recombinant expres- sion vector pET22b-z2151 has been constructed, and the protein z2151 shows good ubiquitin ligase activity. The experiments provide reference for the study of the functional mechanism in the future.

关 键 词：基因工程 肠出血性大肠杆菌O157∶H7 泛素连接酶 体外泛素化 

分 类 号：R378.21[医药卫生—病原生物学] Q78[医药卫生—基础医学]