title: Synthesis and assembly of an adjuvanted Porphyromonas gingivalis fimbrial antigen fusion protein in plants.
abstract: The gram-negative anaerobic oral bacterium Porphyromonas gingivalis initiates periodontal disease by binding to saliva-coated oral surfaces. To assess whether edible plants can synthesize biologically active P. gingivalis fimbrial antigen, for application as an oral vaccine, a cDNA fragment encoding the C-terminal binding portion of P. gingivalis fimbrial protein (FimA), was cloned into a plant expression vector immediately downstream of a cDNA fragment encoding the cholera toxin B subunit (CTB). The chimeric plasmid was transferred into potato (Solanum tuberosum) cells and the ctb-fimA cDNA fragment detected in transformed leaf genomic DNA by PCR amplification methods. A novel protein band of 21 kDa was detected in transformed potato tuber extracts by immunoblot analysis. Oligomeric CTB-FimA (266-337) fusion protein was identified in the extracts through the binding of anti-CTX and anti-native fimbriae antibodies. The pentameric structure of CTB-FimA fusion protein was confirmed by ELISA measurements of GM1 ganglioside receptor binding. Quantification of the CTB-FimA fusion protein by ELISA indicated that the chimeric protein made up about 0.33% of total soluble tuber protein. The biosynthesis of immunologically detectable CTB-FimA fusion proteins and the assembly of fusion protein monomers into biologically active pentamers in transformed potato tuber tissues demonstrate the feasibility of synthesizing adjuvanted fimbrial protein in edible plants for development of adjuvanted mucosal vaccines against P. gingivalis generated periodontal disease.
authors.affiliation: Department of Biochemistry and Microbiology, Center for Molecular Biology and Gene Therapy, School of Medicine, Loma Linda University, Loma Linda, CA 92350, USA.
authors.forename: Eun-Ah
authors.initials: EA
authors.lastname: Shin
authors.forename: Jin-Yong
authors.initials: JY
authors.lastname: Lee
authors.forename: Tae-Geum
authors.initials: TG
authors.lastname: Kim
authors.forename: Yong Keun
authors.initials: YK
authors.lastname: Park
authors.forename: William H R
authors.initials: WH
authors.lastname: Langridge
mesh_terms: D000276:Adjuvants, Immunologic / Q000008:administration & dosage / Q000138:chemical synthesis / Q000235:genetics* / Q000378:metabolism; D000942:Antigens, Bacterial / Q000008:administration & dosage / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D016866:Bacteroidaceae Infections / Q000382:microbiology / Q000517:prevention & control; D002772:Cholera Toxin / Q000008:administration & dosage / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D036781:Fimbriae Proteins / Q000008:administration & dosage / Q000138:chemical synthesis* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D010510:Periodontal Diseases / Q000382:microbiology / Q000517:prevention & control; D030821:Plants, Genetically Modified; D016966:Porphyromonas gingivalis / Q000235:genetics* / Q000276:immunology; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000138:chemical synthesis* / Q000235:genetics / Q000378:metabolism; D011198:Solanum tuberosum / Q000235:genetics / Q000378:metabolism; D022223:Vaccines, Subunit / Q000008:administration & dosage / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
chemical_list: D000276:Adjuvants, Immunologic; D000942:Antigens, Bacterial; D011993:Recombinant Fusion Proteins; D022223:Vaccines, Subunit; C063055:fimbrillin; D036781:Fimbriae Proteins; D002772:Cholera Toxin