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Recombination and Expression of eaeA Gene in Enterohemorrhagic Escherichia coli O157:H7

Kim, Hong   (Department of Biomedical Laboratory Science, College of Health Science, Yonsei UniversityUU0000936  ); Kim, Jong-Bae   (Department of Biomedical Laboratory Science, College of Health Science, Yonsei UniversityUU0000936  );
  • 초록

    Enterohemorrhagic Escherichia coli (EHEC) strains of serotype O157:H7 have been shown to colonize the intestinal epithelial cell by the attaching and effacing (AE) mechanism. The AE lesion is mediated by an intimin, of which production and expression are controlled by a 3-Kb eaeA gene located EHEC chromosomal DNA. If the eaeA gene is mutated, EHEC O157:H7 strains lose capacity of adhesion to intestinal epithelial cells. In this study, a 891 bp of the 3'-end region of a gamma intimin was amplified by polymerase chain reaction (PCR). The PCR product was inserted into pSTBlue-1 cloning vector and transformed into DE3 (BL21) competent cell. After plasmid mini-preparation and restriction enzyme digestion of eaeA/891-pSTBlue-1 vector, target eaeA gene was re-inserted into pET-28a expression vector and was transformed. Then the expression of recombinant eaeA/891 (891 bp) gene was induced by isopropyl- $\beta$ -D-thiogalactopyranoside (IPTG). The expression of the 40-KDa recombinant protein was identified in SDS-PAGE and confirmed by immunoblotting using the His.Tag $^{\circledR}$ and T $_{7}$ .Tag $^{\circledR}$ monoclonal antibody. This recombinant protein expressed by eaeA gene could be applied in further studies on the mechanisms of E. coli O157:H7 infection and the development of recombinant vaccine.


  • 주제어

    EHEC O157:H7 .   Intimin .   eaeA gene .   PCR .   Immunoblotting.  

 저자의 다른 논문

  • Kim, Hong (5)

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    2. 2000 "다중 중합효소 연쇄반응을 이용한 소의 Johne병 진단 기법 확립" The Journal of biomedical laboratory sciences = 대한의생명과학회지 6 (1): 65~72    
    3. 2000 "A Novel PCR Primers HPU185 and HPL826 Based on 16S rRNA Gene for Detection of Helicobacter pylori" The journal of the Korean Society for Microbiology 35 (4): 283~288    
    4. 2001 "Comparison of Different PCR-Based Genotyping Techniques for MRSA Discrimination Among Methicillin-Resistant Staphylococcus aureus Isolates" Journal of microbiology and biotechnology 11 (5): 788~797    
    5. 2001 "Detection of Enterotoxins in Staphylococcus aureus Isolated from Clinical Specimens and Kimbap Using Multiplex PCR" The Journal of biomedical laboratory sciences = 대한의생명과학회지 7 (2): 85~89    
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