| MOLECULAR CLONING AND SEQUENCE ANALYSIS OF THE GENE FOR THE HEMIN-BINDING PROTEIN FROM Prevotella intermedia |
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| Prevotella intermedia에서의 Hemin 결합 단백질 유전자의 분리 및 염기서열 분석 |
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김신1, 김성조2 |
1부산대학교 치과대학 소아치과학교실
2부산대학교 치과대학 치주과학교실 |
Correspondence:
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| Abstract |
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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 heminbinding 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 phonotype. The 2.5kb 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. |
| Key Words:
Hemin, Hemin-binding protein, Prevotella intermedia, Gene cloning |
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