1999 Fiscal Year Final Research Report Summary
Expression of the gene coding for Staohylococcus hyicus exfoliative toxin in Escherichia coli
| Project/Area Number |
10660292
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Basic veterinary science/Basic zootechnical science
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| Research Institution | Kitasato University |
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Principal Investigator |
MAEHARA Nobutoshi Kitasato Univerisity, School of Veterinary Medicine and Animal Science, Professor, 獣医畜産学部, 教授 (90072371)
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| Co-Investigator(Kenkyū-buntansha) |
SATO Hisaaki Kitasato Univerisity, School of Veterinary Medicine and Animal Science, Assistant Professor, 獣医畜産学部, 講師 (40154083)
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| Project Period (FY) |
1998 – 1999
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| Keywords | Staphylococcus hyicus / Exfoliative toxin / Expression / Gene / Escherichia coli |
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| Research Abstract |
Staphylococcus hyicus exfoliative toxin B (SHETB) gene and Staphylococcus aureus exfoliative toxin C (ETC) gene were amplified by polymerase chain reaction, cloned in expression vector pET-32a and transformed to Escherichia coli strain BL2 l. Then each transformant was tested for the expression of each toxin (SHETB and ETC) by SDS-polyacrylamide gel electrophoresis. Results from electrophoretic analysis of each expressed protein (SHETB and ETC) revealed the presence of a prominently stained band corresponding to a molecular weight of 47 kDa, The bands are in agreement with the predicted size from the DNA sequence of genes coding for SHETB and ETC. The recombinant SHETB and ETC proteins contained a amino terminal histidine were purified by the nickel chelate affinity chromatography. The eluted expressed SHETB proteins caused exfoliation in 1-day-old chicken and the eluted expressed ETC proteins caused exfoliation in both 1-day-old chicken and suckling mice within 1 h. In western blot analysis by use of monoclonal antibody against each toxin, recombinant SHETB (r-SHETB) gave a single band (47 kDa) to anti-SHETB antibody alone and that recombinant ETC (r-ETC) gave a single band (47 kDa) to anti-ETC antibody alone, These results indicated that biological activity and antigenicity of recombinant proteins are coincide with original toxin proteins of SHETB and ETC. The yields of r-SHETB and r-ETC were 75 and 10 times higher than those of SHETB from S. hyicus and ETC from S. aureus. These results suggest that these recombinant toxins were useful for the analysis of the correlation between molecular composition and biological activity of these toxins.
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