[Construction of eukaryotic expression plasmid pcDNA3-gtfB expressing glucosyltransferase B of Streptococcus mutans].
OBJECTIVE
Glucosyltransferase (GTF) of Streptococcus mutans is considered as a cariogenic virulence factor due to its ability to synthesize glucan, which facilitate sucrose-depended adherence and cell-to-cell accumulation of bacteria. In this study, gtfB, the target gene fragment which encodes multiple catalytic sites and antigen epitopes of GTF, was recombined into eukaryotic expression vector pcDNA3. The feasibility of the recombination plasmid pcDNA3-gtfB used as gene vaccine will be investigated in further study.


METHODS
The target gene fragment gtfB (904-4578 bp) was obtained by standard PCR amplification while genome DNA of streptococcus mutans GS-5 was used as template. Then the PCR products were extracted and purified from low-melting temperature agarose. The gtfB and plasmid pcDNA3 were cut by Kpn I, Xho I, and the digested products were extracted and purified again for recombination. The purified gtfB and plasmid pcDNA3 were recombined by T4 DNA ligase, ligation products were transformed into competent cell, Escherichia coli JM109. Transformed colonies were screened by Ampr LB plate, then recombined plasmids were isolated and identified by restricted endonuclease cutting and Sanger dideoxy DNA sequencing.


RESULTS
Identified by agarose gel electrophoresis, the target gene-gtfB obtained PCR amplification had the same molecular size (36 kb) as predicted. It was indicated that recombined plasmids contained inserted gtfB gene fragment by restricted endonuclease cut analysis, the sequencing data also indicated that inserted gtfB gene had correct DNA sequence and orientation according to DNA sequence of Streptococcus mutans GS-5 (gene bank M17361).


CONCLUSIONS
Inserted gene-gtfB of recombined plasmid pcDNA3-gtfB encoded multiple catalytic sites and epitopes. It was proved that these epitopes had high immune antigenicity and that antiserum could significantly inhibit the synthesis of water-insoluble glucans and water-soluble glucan. In vitro adherence experiment also indicated that it could inhibit streptococcus mutans adherence to saliva-coated hydroxyapatite. Vector pcDNA3 was high expressing eukaryotic vector, and could stimulate antigen-representing cell. It was suggested that recombined plasmid pcDNA3-gtfB had high immune antigenicity and immune responsiveness, and this supported its use as gene vaccine candidates in the development of anti-caries vaccines.
Yang
J
J
College of Stomatology, West China University of Medical Sciences.
Liu
T
T
Zhuo
X
X
D003731:Dental Caries / Q000517:prevention & control; D005057:Eukaryotic Cells / Q000378:metabolism*; D005822:Genetic Vectors; D005964:Glucosyltransferases / Q000235:genetics*; D010957:Plasmids / Q000235:genetics* / Q000276:immunology; D011995:Recombination, Genetic; D013295:Streptococcus mutans / Q000235:genetics*; D019444:Vaccines, DNA
D019444:Vaccines, DNA; D005964:Glucosyltransferases