• 대한수의학회지
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• 제44권2호
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• pp.251-257
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• 2004

This study was designed to determine optimal condition for expression of cloned Shigatoxin2e(Stx2e) gene from transformed E. coli PED18, to compare the cytotoxicity titer between cloned Stx2e and Stx2e from original strain, and to confirm of receptor binding affinity of Stx2e for use of development of receptor binding ELISA to detect of Stx2e. The optimum composition of medium for expression of Stx2e gene in E.coli host-vector system was definded as the medium containing 0.5% glucose and 0.5 mM IPTG. The cytotoxicity titer of expressed Stx2e for Vero cell was 1000 fold higher than that of Stx2e from original strain AY93258. The binding affinity of Stx2e to receptor globotetraosyl ceramide($Gb_4$) was confirmed by immunobloting.

• 대한수의학회지
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• 제44권2호
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• pp.233-239
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• 2004

This study was designed to quantitative assay of cellular receptor globotriosyl ceramide($Gb_3$) and globotetraosyl ceramide($Gb_4$) and to determine binding affinity of the receptors for Shigatoxin 2e in difference with day olds of pig. The amount of $Gb_3$ and $Gb_4$ extracted from each swine tissue were detected in order of spleen, ileocecum, kidney, and jejunum. The amount of $Gb_3$ and $Gb_4$ extracted from each swine ileocecum and jejunum in different day old swine were detected in order of 24, 6, 98, 12 and 35, but the difference of amount was not significant. The binding affinity of $Gb_4$ extracted from ileocecum and jejunum with different day old swine for Stx2e were shown that the extracts of 24, 12, and 35 days old swine were higher significantly than that of 6 and 98 days old swine.

• 한국임상수의학회지
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• 제22권4호
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• pp.386-391
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• 2005

This study was done to produce a mutated protein inactivated cytotoxicity of Shigatoxin 2e (Stx2e) of E.coli O139 isolates by deletional mutagenesis of Stx2e A subunit gene encoding active-site cleft of enzymatic domain in ST2e holotoxin. Cytotoxicity of the toxoid expressed from the mutant Stx2e gene was compared with wild type Stx2e for development of vaccine candidate. A recombinant plasmid pED18 containing Stx2e gene ot E.coli O139 isolates was used to generate mutation plasmid. Deletion mutagenesis was conducted for Stx2e A subunit gene encoding enzymatically active domain by polymerase chain reaction (PCR) using ot designed primer to induce deletional mutation. DNA sequence analysis was confirmed that the pentamer (Typ 202- Ser 206) that lies within the proposed active-site cleft in the second region was completely deleted. A DNA fragment of 1.1 kb that encode the new mutant Stx2eA gene was inserted into plasmid pRSET vector digested with EcoRV-Hind III and named pEDSET The PEDSET was transformed in E. coli for expression of mutant protein and the protein was confirmed by SDS-PACE and Western-blotting. The protein expressed by the mutant was tested to confirm the reduction of cytotoxic activities on Vero cell using microcytotoxicity assay compared with wild type Stx2e, the cytotoxicity of deletional mutant protein was at least reduced by 3,000-fold on Vero cell.