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当前位置: 首页 > 文章 > 大肠杆菌O157:H7候选效应蛋白Z1370的原核表达、纯化及多抗制备 中国兽医学报 2015,35 (12) 1959-1964
Position: Home > Articles > Prokaryotic expression,purification and qualified polyclonal antibody preparation of Escherichia coli O157: H7 candidate effector Z1370 Chinese Journal of Veterinary Science 2015,35 (12) 1959-1964

大肠杆菌O157:H7候选效应蛋白Z1370的原核表达、纯化及多抗制备

作  者：
王豪举;闫青;毛旭虎;丁红雷
单  位：
西南大学动物科技学院;中国农业大学动物医学院;第三军医大学西南医院医学检验系
关键词：
肠出血性大肠杆菌O157:H7;Z1370;分泌蛋白;多克隆抗体;原核表达
摘  要：
为鉴定肠出血性大肠杆菌O157:H7的效应蛋白,本研究利用生物信息学方法预测了1个新的大肠杆菌O157:H7分泌蛋白Z1370,并以EDL 933菌株为模板,扩增Z1370基因。将该基因克隆至原核表达载体p GEX-6P-2,转化大肠杆菌XL-1 Blue,IPTG诱导目的蛋白表达。利用谷胱甘肽琼脂糖珠和Pre Scission Protease酶纯化目的蛋白。纯化的目的蛋白免疫小鼠制备多抗血清并鉴定多抗血清的免疫反应性和特异性。结果表明,Z1370可能是一个能够分泌至细胞外的效应蛋白。将PCR扩增的Z1370基因连接表达载体并成功在大肠杆菌以可溶形式表达了具有良好天然构象的蛋白,该蛋白在大肠杆菌O157:H7中可以自身催化,并以小于原始相对分子质量的形式存在。将该蛋白纯化后免疫小鼠制备了具有良好免疫反应性和特异性的多抗血清。这为Z1370蛋白的亚细胞定位和功能研究奠定了基础、提供了研究工具。
译  名：
Prokaryotic expression,purification and qualified polyclonal antibody preparation of Escherichia coli O157: H7 candidate effector Z1370
作  者：
WANG Hao-ju;YAN Qing;MAO Xu-hu;DING Hong-lei;College of Animal Science and Technology,Southwest University;College of Veterinary Medicine,China Agricultural University;Department of Laboratory Medicine,Southwest Hospital,Third Military Medical University;
关键词：
enterohemorrhagic Escherichia coli(EHEC) O157;; H7;;Z1370;;secretory protein;;polyclonal antibody;;prokaryotic expression
摘  要：
In order to identify the effector protein of enterohemorrhagic Escherichia coli( EHEC) O157:H7,a new secretory protein called Z1370 was predicted by bioinformatics analysis. Z1370 was amplified by using EDL 933 genome as the template and cloned into prokaryotic expression vector p GEX-6P-2. The recombinant vector was transformed into E. coli XL-1 Blue,and the Z1370 protein was expressed by IPTG inducing,and purified by Glutathione SepharoseTM4 B. Polyclonal antibody against Z1370 was acquired by immunization of mice,and the immunoreactivity and specificity of the antisera was evaluated. The bioinformatics information result showed that Z1370 could be secreted by E. coli O157: H7,which maybe is as an effector protein. Z1370 was amplified by PCR,and ligated into p GEX-6P-2,and then the gene was solublly expressed by E. coli XL-1 Blue in a native conformation. Interestingly,the protein was autocatalyzed and existed as a less molecular weight form. The antisera acquired from immunized mice with purified Z1370 have good immunoreactivity and specificity. This research established a foundation for the subcellular localization and function research of Z1370.

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