Prokaryotic expression of Tir gene of enterohemorrhagic escherichia coli
YU Fang
LONG Bei-guo
WANG Jin-hui
WANG Rui-ning
WU Xian-bo
FAN Hong-ying
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.
Keywords：EHEC O157:H7Tir-CExpressionActivity identification
Publication Date：2014-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：4( 1519-1522 )