Prokaryotic expression of Tir gene of enterohemorrhagic escherichia coli
10.3969/j.issn.1006-5725.2014.10.002
- VernacularTitle:肠出血型大肠埃希菌O157：H7 Tir基因的原核表达
- Author:
Fang YU
;
Beiguo LONG
;
Jinhui WANG
;
Ruining WANG
;
Xianbo WU
;
Hongying FAN
- Publication Type:Journal Article
- Keywords:
EHEC O157:H7;
Tir-C;
Expression;
Activity identification
- From:
The Journal of Practical Medicine
2014;(10):1519-1522
- CountryChina
- Language:Chinese
-
Abstract:
Objective To identify the immune activity of the recombinant protein Preli minarily after expressing Tir C-ter minal of enterohemorrhagic Escherichia coli(EHEC) in E.coli BL21/DE3 efficiently. Methods Tir C-ter minal (marked as Tir-C)was amplified by PCR considering the result of Bioinformatics analysis of Tir. The recombinant plasmid(designed as PET-30a(+)-Tir-C)was identified by PCR,sequencing and digested by restriction endonucleases. The positive recombinants were transformed into E.coli BL21/DE3 and induced by IPTG to express the Tir-C. The Tir-C protein was detected by SDS-PAGE and identify the antigenic of the recombinant protein Preli minarily by Western blot. Results The 675bp DNA was gained. The plasmid PET-30a (+)-Tir-C was built. Tir-C was expressed mainly in supernatant of lysis and was purified by Ni+affinity chromatograghy. Concentration of the purified protein is about 500 μg/mL and a unique band was detected with the relative molecular mass of approximately 24KDa by Western blot. Conclusion The recombinant Tir-C was expressed successfully and had immunoreactivity to some extent, which deserves the investigations for vaccine against EHEC.
