[Expression and identification of enterohemorrhagic Escherichia coli O157:H7 Shiga toxin1A subunit].
OBJECTIVE
To express and identify enterohemorrhagic Escherichia coli (EHEC) O157:H7 Shiga toxin 1 A subunit (Stx1A).


METHODS
Stx1A encoded gene was amplified from EHEC O157:H7 genome by PCR, confirmed by sequencing and cloned into vector pET-22b(+). The recombinant plasmid pET-22b(+)-Stx1A was transformed into E.coli BL21(DE3) which was induced by IPTG to express the target protein. After purified by AKTA(TM);-His affinity chromatography, the recombinant protein was identified by mass spectrometry. With the recombinant protein, BALB/c mice were immunized to develop the anti-sera and evaluate its specific reaction with the natural Stx1A by Western blotting.


RESULTS
The Stx1A gene with a size of 945 bp was amplified and cloned into prokaryotic expression vector pET22b(+) to form pET-22b(+)-Stx1A. The recombinant protein was effectively expressed in E.coli BL21(DE3) and purified by 6×His-based affinity chromatography. The mass spectrometry analysis showed that the target protein was Stx1A. Western blotting demonstrated that its immunized sera could react specifically with the natural Stx1A.


CONCLUSIONS
The EHEC O157:H7 Stx1A gene was successfully cloned and expressed, which laid a solid foundation for the following researches.
Veterinary School, Nanjing Agricultural University, Nanjing 210095, China.
Mingming
M
Huang
Institute of Pathogenic Microbiology, Jiangsu Provincial Center for Disease Prevention and Control, Nanjing 210009, China.
Xiaoyan
X
Zeng
Institute of Pathogenic Microbiology, Jiangsu Provincial Center for Disease Prevention and Control, Nanjing 210009, China.
Fengjuan
F
Shi
Institute of Pathogenic Microbiology, Jiangsu Provincial Center for Disease Prevention and Control, Nanjing 210009, China.
Li
L
Zhang
Veterinary School, Nanjing Agricultural University, Nanjing 210095, China.
Xiangrui
X
Li
Institute of Pathogenic Microbiology, Jiangsu Provincial Center for Disease Prevention and Control, Nanjing 210009, China.
Yongjun
Y
Jiao
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D019453:Escherichia coli O157 / Q000235:genetics*; D005260:Female; D015870:Gene Expression; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D022622:Shiga Toxin 1 / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification
D021122:Protein Subunits; D022622:Shiga Toxin 1