Prevotella intermedia에서의 Hemin 결합 단백질 유전자의분리 및 염기서열 분석
Molecular cloning and sequence analysis of the genefor the hemin-bonding protein from prevotella intemedia
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본 연구는 치주질환 주요 병인균주 중의 하나인 P. intermedia를 대상으로 하여, 이 균주에서의 hemin 결합 단백질 유전자를 분리하고 염기서열을 결정하기 위하여 수행되었다. 본 연구에서는 약 5,000개의 recombinant colony들을 스크리닝하여 hemin 결합 단백질 유전자를 포함하고 있는 것으로 여겨지는 1개의 클론 (pHem1)을 확인하였다. Restriction enzyme mapping 결과 pHem1의 insert DNA의 크기는 약 2.5kb이었으며, P. intermedia chromosomal DNA 내에는 hemin 결합 단백질 유전자가 single copy로 존재하였고, transcript의 크기는 약 1.8kb 이었다. 분리한 pHem1 유전자는 1개의 ORF를 가지고 있으며, ORF의 크기는 2,550bp로 약 850개의 아미노산 폴리펩타이드로 구성되어 있다. 또한, pHem1 유전자는 이미 밝혀진 다른 유전자들과 상동성을 보이지 않았다. 본 연구는 P. intermedia에서의 porphyrin 생리 및 hemin 획득기전을 분자생물학적으로 규명하는데 있어 중요한 의의가 있으리라 사료된다. 향후 pHem1 유전자의 특성 분석 등이 수행되어야 할 것으로 여겨진다.
Abstract
Prevotella intermedia is one of the most frequently implicated pathogens in human periodontal disease and has a requirement for hemin for growth. This study has identified a hemin-binding P. intermedia protein by expression of a P. intermedia genomic library in Escherichia coli, a bacterium which does not require or transport exogenous hemin. The genomic library of P. intermedia was constructed into plasmid pUC18, transformed into Escherichia coli strain DH5α, and screened for recombinant clones using hemin-binding activity by plating onto hemin-containing agar. Approximately 5,000 recombinant E. coli colonies were screened onto LB-amp-hemin agar, single clone (pHem1) was exhibited a clearly pigmented phenotype. The 2.5 kb insert DNA of pHem1 was determined by restriction enzyme mapping. Southern blot analysis of BamHI, BglII, EcoRI, HindIII and PstI-digested P. intermedia DNA indicated that single copy of the gene was present in the genome. Northern blot analysis revealed that the size of transcript was approximately 1.8 kb. The cloned gene contained a single ORF, consisting of approximately 850-residue amino acids. A BLAST search of the Institute for Genomic Research genes with similar nucleotide sequence revealed no significant similarity. It needs further investigation to clarify the mechanisms of heme uptake in P. intermedia.
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