Gene Cloning and Characterization of Short Immunodominant Hemagglutinin A of Porphyromonas gingivalis
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概要
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Porphyromonas gingivalis, a gram-negative anaerobic bacterium, is considered to be one of the major etiologic agents of adult periodontitis. We previously succeeded in the gene cloning of a 200-kDa antigenic protein (200-kDa-Ag) from P. gingivalis 381, designated pMD101, and identified it as the truncated hemagglutinin A (HagA), which was thought to be a useful vaccine and immunodominant antigen for serodiagnosis. HagA has immunodominant regions encoding VTVPENGK in the N-terminal region and four repeats of the functional motif PVQNLT expressing hemagglutination. Unfortunately, the production rate of 200-kDa-Ag was low and inclusion bodies were made in Escherichia coli, so we established a subclone pMD160 harboring in the N-terminal region with two repeats of the functional motif. In the present study, because the maltose binding protein (MBP) is known as an effective adjuvant with a convenient purification process, we attempted to establish a novel gene clone that encodes the minimum immunodominant antigenic region of the N-terminal with a single unit of functional motif PVQNLT for the development of serodiagnosis and vaccination. The DNA nucleotide sequences of the novel subclone, designated as pMD167, revealed that the gene product was composed of the minimum immunodominant antigenic region of HagA and MBP as a fused protein and it was designated as 75-kDa-HagA/MBP. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the pMD167 clone produced higher amounts of recombinant proteins than the pMD101 clone. Western blot analysis showed that the pMD167 recombinant protein was strongly recognized by the sera of periodontitis patients. These findings suggest that the novel recombinant protein 75-kDa-HagA/MBP from pMD167 may be useful for the development of serodiagnosis and immunotherapy against P. gingivalis infection.
著者
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HAYAKAWA Mitsuo
Department of Biochemistry and Molecular Biology, Nihon University School of Dentistry at Matsudo
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Abiko Yoshimitsu
Department Of Biochemistry And Molecular Biochemistry Nihon University School Of Dentistry At Matsud
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Hirai Tomonari
Department of Biochemistry and Molecular Biology, Nihon University School of Dentistry at Matsudo
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