Recombination and expression of eaeA gene in Enterohemorrhagic Escherichia coli O157:H7.

임상병리학과/석사[한글] Enterohemorrahgic Escherichia coli (EHEC) 혈청형 0157:H7은 출혈을 동반한 설사를 일으키고, 1세 미만의 소아에게 감염하여 급성 설사를 유발하며 심한 경우 출혈성 요독증을일으키는 세균으로 알려져 있다. 이 세균은 사람의 장관 내에서 attaching-and-effacing (AE) lesion을 형성하면서 상피세포에 부착하여 집락을 형성한다. AE lesion은 EHEC의chomosomal DNA상에 위치하는 3-Kb의 eaeA 유전자가 발현하는 intimin에 의해서 매개되는데, 이 단백질은 94-KDa의 outer membrane protein (OMP)으로서 eaeA 유전자를 mutation시킬 경우 intimin을 발현하지 못하는 EHEC 0157:H7은 부착능을 잃어버리게 된다. 즉 intimin은 EHEC가 장관 내 상피세포에 부착하는데 중요한 역할을 한다. 본 연구에서는 중합효소 연쇄반응을 이용하여 EHEC 0157:H7에만 독특하게 존재하는 gamma intimin의 3' end에 해당하는 891 bp를 증폭하여 분리하였다. 순수하게 정제한 PCR 증폭 산물을 cloning vector에 삽입하였고, recombinant plasmid를 분리한 후 expression vector에 삽입하여 형질 전환하였다. 그 후 eaeA/891 (891 bp)의 발현을 유도하여, 약 40-KDa의 특이적인 단백질을 확인하였다. 이 단백질은 His·Tag^ⓡ과 T_7·Tag^ⓡ monoclonal antibody를 이용하여 immunoblotting을 실시한 결과 eaeA/891 유전자가 expression vector system에서 발현한 것임을 확인하였다. [영문] Enterohemorrhagic Escherichia coli (EHEC) strains of serotype O157:H7 are the predominant cause of hemorrhagic colitis, an important cause of classical hemolytic uremic syndrom and a major cause of acute infantile diarrhea. In human, these strains have been shown to colonize the intestinal epithelial cell by the attaching and effacing (AE) mechanism. The AE lesion is mediated by a intimin, of which production and expression is controlled by a 3- Kb eaeA gene located EHEC chromosomal DNA. The intimin is a 94- KDa outer membrane protein (OMP). If the eaeA gene is mutated, EHEC O157:H7 strains which can not express the intimin lose capacity .Therefore the intimin plays an important role of binding EHEC strains. In this study, a 891 bp included 3^--end region of a gamma intimin which are only found in EHEC was amplified by polymerase chain reaction(PCR) and was eluted from agarose gel. PCR product eluted clearly was inserted into cloning vector and transformed into DE3(BL21) competent cell provided. After eluted from agarose gel using plasmid mini-preparation and restriction enzyme digestion, PCR product was re-inserted into expression vector and was transformed. Then for expression eaeA/891 (891 bp) was induced by isopropyl-β-D-thiogalactopyranoside(IPTG). The expression of the 40- KDa specific recombinant protein was identified in SDS-PAGE and the protein was originated from recombinant eaeA/891 gene in expression vector system confirmed by immunoblotting using the His. Tag^ⓡ and T_7ㆍTag^ⓡ monoclonal antibody. The protein expressed by eaeA gene isolated EHEC O157:H7 could be applied in the production of polyclonal antibody and development of recombinant vaccine.ope