Prokaryotic expression, purification, identification, and polyclonal antibody prepa-ration of enterohemorrhagic Escherichia coli effector NleB1
10.7644/j.issn.1674-9960.2014.10.013
- VernacularTitle:肠出血性大肠杆菌O157∶H7毒力蛋白NleB1的表达、纯化及多克隆抗体的制备与鉴定
- Author:
Xinxin CHEN
;
Xiang LIAO
;
Ting SONG
;
Wei ZHOU
;
Hongmei DAI
;
Junjie YUE
;
Yu WANG
;
Yurui WANG
;
Long LIANG
- Publication Type:Journal Article
- Keywords:
enterohemorrhagic Escherichia coli;
NleB1;
prokaryotic expression;
protein purification;
polyclonal antibody
- From:
Military Medical Sciences
2014;(10):799-802
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a prokaryotic plasmid expressing the recombinant protein of enterohemorrhagic Escherichia coli(EHEC) effector NleB1 and to prepare the polyclonal antibody of mouse anti-NleB1.Methods The nleB1 (990 bp) gene was amplified from the genome EHEC O157∶H7 and cloned into the expression plasmid pET24a to construct the recombinant plasmid pET24a-nleB1 that was transformed into E.coli BL21(DE3).After induction with isopropylthio-gelactoside( IPTG) , the His-tag fusion proteins were purified by Ni+affinity chromatography and gel slices.The polyclonal antibody was prepared by immunizing BALB/c mice with purified recombinant proteins and analyzed by Western blotting and ELISA.Results The pET24a-nleB1 recombinant plasmid was successfully constructed, the fusion protein was ex-pressed and purified,and the polyclonal antibody was obtained by immunizing mice with purified fusion protein.Western blotting and ELISA staining demonstrated that the polyclonal antibody was successfully obtained.Conclusion The prepara-tion of the polyclonal antibody against EHEC O157∶H7 NleB1 will be of help for further studies on the function of NleB1 protein.
