关键词: 龋病, 变形链球菌, 葡糖基转移酶, 基因重组, 基因疫苗

Abstract:

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 fragmentwhich encodes multiple catalytic sites and antigen epitopes of GTF, was recombined into eukaryotic ex- pression 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 (904-4578bp) was obtained by standard PCR amplification while genome DNAof 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 KpnÑ, XhoÑ, 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 colonieswere screened by AmprLB plate, then recom- bined plasmidswere isolated and identified by restricted endonuclease cutting and Sangerdideoxy DNAsequencing.Results:Identi- fied by agarose gel electrophoresis, the target gene-gtfB obtained PCR amplification had the same molecular size (36kb) as pre- dicted. Itwas indicated that recombined plasmids contained inserted gtfB gene fragment by restricted endonuclease cut analysis, the sequencing data also indicated thatinserted gtfB gene had correctDNA sequence and orientation according to DNA sequence of Streptococcus mutans GS-5(gene bank M17361).Conclusion:Inserted gene-gtfB of recombined plasmid pcDNA3-gtfB encoded multiple catalytic sites and epitopes. Itwas 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 streptococcusmutans adherence to saliva-coated hydroxyapatite. Vector pcDNA3 was high expressing eukaryotic vector, and could stimulate antigen-representing cell. Itwas 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.

Key words: caries, Streptococcus mutans, glucosyltransferase, gene recombination, gene vaccine