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Construction and Immunogenic Characterization of a Fusion Anti-caries DNA Vaccine against PAc and Glucosyltransferase I of Streptococcus mutans

J.H. Guo, R. Jia, M.W. Fan^*, Z. Bian, Z. Chen, and B. Peng

Key Lab. for Oral Biomedical Engineering of Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan 430079, PR China;

View larger version (29K): [in a new window]   Figure 1. Construction and verification of plasmid pGLUA-P. (A) The GLU region of GTF-I of S. mutans was cloned into the eukaryotic expression plasmid pCIA-P to obtain the recombinant plasmid pGLUA-P. (B) Restriction enzyme digestion of pGLUA-P and pCI. pGLUA-P, A-P fragment, GLU fragment, and pCI were measured as 7.1 kb, 2.2 kb, 0.9 kb, and 4.0 kb, respectively. Lane 1: GeneRuler 1 kb DNA ladder. Lane 2: pGLUA-P digested by SmaI. Lane 3: pCI digested by SalI. Lane 4: pGLUA-P digested by SalI and XhoI. Lane 5: pGLUA-P digested by SalI. Lane 6: DNA/HindIII marker.

View larger version (339K): [in a new window]   Figure 2. Recombinant fusion protein could be expressed in eukaryotic cells. (A) Recombinant fusion protein detected by anti-GTF antibody in the cytoplasm of CHO cells transfected with pGLUA-P. (B) Recombinant fusion protein detected by anti-PAc antibody in the cytoplasm of CHO cells transfected with pGLUA-P. (C) CHO cells transfected with pCIA-P, detected by anti-PAc antibody. (D) CHO cells transfected with pCI vector. Bar, 5 µm.

View larger version (20K): [in a new window]   Figure 3 Antibody levels and caries scores of rats immunized with various treatments. Data are expressed as means ± standard deviation. Groups of rats were immunized with plasmids as follows: intramuscular injection of pGLUA-P (GLUA-P/i.m.); subcutaneous injection near the submandibular gland of pGLUA-P (GLUA-P/s.c.); intramuscular injection of pCIA-P (A-P/i.m.); subcutaneous injection of pCIA-P (A-P/s.c.) near the submandibular gland; and intramuscular injection of pCI (control). *Significantly different from the control group at p < 0.01; **significantly different from the control and pCIA-P groups at p < 0.01; #significantly different from DNA-vaccine-immunized group via i.m. at p < 0.01; ^significantly different from DNA-vaccine-immunized group via s.c. at p < 0.05; and +significantly different from pCIA-P immunized group via s.c. at p < 0.05. (A) Saliva- and serum-specific anti-PAc antibody levels (five rats per group). Serum anti-PAc IgG; salivary anti-PAc IgA. (B) Saliva- and serum-specific anti-GTF antibody levels (five rats per group). • Serum anti-GTF IgG; salivary anti-GTF IgA. (C) Keyes caries scores (five rats per group). • Enamel lesion; slight dentinal lesion; moderate dentinal lesion.