Subcloning of the 200-kDa Porphyromonas gingivalis antigen gene and inhibition of hemagglutination by an antibody against the recombinant protein.
Porphyromonas gingivalis is a major etiologic agent of periodontitis and exhibits hemagglutinating and adherence activities. We previously succeeded in molecular cloning the 200-kDa cell-surface antigenic protein (200-k AP), designated pMD101, that is recognized in sera from periodontitis patients, and identified the 200-k AP as a hemagglutinin A (HagA) derivative. HagA is one of the hemagglutinins known to be a useful vaccine against periodontitis. HagA has four large, contiguous, direct repeats and the repeat unit is believed to contain the hemagglutinin domain. Because production of 200-k AP was low in the Escherichia coli host, it was difficult to obtain large amounts of recombinant protein. In this study, we attempt to subclone the gene encoding the useful antigen from pMD101 in an effort to obtain large quantities. A subclone, designated pMD160, encoding a fusion protein of 80-kDa HagA and maltose-binding protein was successfully constructed, and the novel clone produced relatively large amounts of recombinant protein. DNA nucleotide sequences of the pMD160 insert demonstrated that the 80-kDa protein contained a short hemagglutinin motif and a direct repeat unit region. The recombinant protein was purified to homogeneity and rabbit antiserum was raised. The antibody was capable of inhibiting the hemagglutinating activity of P. gingivalis. These findings suggest that novel 80-kDa HagA derivative proteins can be produced efficiently from E. coli hosts and these may be useful in developing immunotherapy against periodontitis infected by P. gingivalis.
Suyama
Tsutomu
T
Departments of Biochemistry, Nihon University School of Dentistry at Matsudo, Chiba, Japan.
Hayakawa
Mitsuo
M
Abiko
Yoshimitsu
Y
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology; D006384:Hemagglutination / Q000235:genetics; D006388:Hemagglutinins / Q000235:genetics* / Q000276:immunology; D037102:Lectins; D008969:Molecular Sequence Data; D010518:Periodontitis / Q000382:microbiology*; D016966:Porphyromonas gingivalis / Q000235:genetics* / Q000276:immunology; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics
D001426:Bacterial Proteins; D006388:Hemagglutinins; D037102:Lectins; D011994:Recombinant Proteins; C114923:hemagglutinin A, Porphyromonas gingivalis