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KMID : 0545120030130040552
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Journal of Microbiology and Biotechnology
2003 Volume.13 No. 4 p.552 ~ p.559
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Expression and Characterization of Uropathogenic Escherichia coli Adhesin Protein Linked to Cholera Toxin A2B Subunits in Escherichia coli TB1
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LEE, YONG-HWA
RYU, DONG-KYUNG/KIM, BYUNG-OH/PYO, SUHKNEUNG
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Abstract
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The FimH subunit of type I-fimbriated E.ycherichia coli (E. roli) has been determined as a major cause for urinary tract infections.
Thus, to produce a possible vaccine antigen against urinary tract infections.
the fimH gene was genctically coupled to the ctxa2b gene and cloned into a pMAL-p2E expression vector. The chimeric construction of pMALfimHl ctxu2b was then transformed into E. coli K-12 TBI and its nucleotide sequence was verified.
A fusion protein, based on fusing adhesin to the cholera toxin subunit A2B (CTXA2B), was induced with 0.01 mM isopropyl-β-D-thiogalactoside (TPTG) for 4 h at 37℃ to yield a soluble fusion protein. The fusion protein was then purified by affinity chromatography. The expressed fusion protein was confirmed by SDS-PAGE and Westem blotting using antibodies to the maltose binding protein (MBP) or the cholera toxin subunit B (CTXB), plus the N-terminal amino acid sequence was also analyzed. The orderly-assembled fusion protein was confirmed by a modified G_(m?),-ganglioside ELISA, using antibodies to adhesin.
The results indicated that the purified lusion protein was an adhesin/CTXA2B protein containing E. coli adhesin and the G,,-ganglioside binding activity of CTXB. Accordingly, this adhesin/CTXA2B protein may he a potential antigen for oral immunization against uropathogenic E. coil .
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