title: Construction of a functional single-chain variable fragment antibody against hemagglutinin from Porphyromonas gingivalis.
abstract: Hemagglutinin is a major glycoprotein of Porphyromonas gingivalis vesicles and likely confers the ability to adsorb and penetrate into host tissue cells. To protect this bacterial invasion, murine monoclonal antibody (MAb) Pg-vc, which inhibited the hemagglutinating activity, was prepared by using P. gingivalis vesicles as an antigen. Western blot analysis revealed that when both MAb Pg-vc and anti-HA-Ag2 antibody raised against the P. gingivalis hemagglutinin adhesin (M. Deslauriers and C. Mouton, Infect. Immun. 60:2791-2799, 1992) were allowed to react with protein blots from P. gingivalis vesicles, a superimposable profile was observed. To obtain a recombinant antibody, cDNAs coding for the variable domains of the L and H chains of MAb Pg-vc were cloned by PCR, and a plasmid specifying a single-chain variable fragment (ScFv) was constructed. Following transformation of Escherichia coli cells, a recombinant ScFv protein was successfully expressed. The immunological properties of this protein were identical to those of the parental murine MAb, specifically recognizing the two proteins (43 and 49 kDa) originating from P. gingivalis vesicles. In addition, the ScFv antibody inhibited the P. gingivalis vesicle-associated hemagglutinating activity. The amino acid sequences deduced from nucleotide sequencing experiments confirmed that variable heavy-chain and variable light-chain regions belonged to VH1 and Vkappa12/13 families, respectively. Since the expression system used in this study can readily provide large quantities of single-chain recombinant antibody, it may be a useful in developing a therapeutic agent for passive immunization in humans.
authors.affiliation: Department of Biochemistry, Nihon University School of Dentistry at Matsudo, Chiba, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Shibata
authors.forename: K
authors.initials: K
authors.lastname: Kurihara
authors.forename: H
authors.initials: H
authors.lastname: Takiguchi
authors.forename: Y
authors.initials: Y
authors.lastname: Abiko
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000737:chemistry / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001483:Base Sequence; D006388:Hemagglutinins / Q000276:immunology*; D007116:Immunization, Passive; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016966:Porphyromonas gingivalis / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology
chemical_list: D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D006388:Hemagglutinins; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; C055966:immunoglobulin Fv