[Construction and expression of traceable DNA vaccine for prevention of caries].
OBJECTIVE
Streptococcus mutans has been proved as a causative bacteria of human dental caries. The surface protein antigen is one of the important pathogenic factors. The A region of the surface protein antigen pac gene (pacA) can enrich T-cells and B-cells epitope. In this study, a DNA vaccine carrying pacA and gfp gene (a reporter gene) for caries prevention was constructed. The DNA vaccine was liable to be traced in vitro and in vivo.


METHODS
The fragment of pacA (1.3 kb) was amplified by PCR with the plasmid pPC41 as template, and inserted into a pEGFP-C1 vector. The recombinant plasmid produced was named as pEGFPC1-pacA. After the COS1 cell line was transfected by the recombinant plasmid, the expression of gfp was detected by observing the green fluorescence and measuring the fluorescence intensity, and the expression of pacA was detected by RT-PCR.


RESULTS
Restricted analyzing, sequencing and PCR technique were employed to identify the recombinant plasmid. The phase and orientation of the pacA gene inserted into the vector pEGFPC1 were correct and no changes of their open reading frames were discovered. The transfected COS1 carrying green fluorescent protein (GFP) was observed; the GFP expression level of transfected cells was higher than that of controlled cell. The transcript of pacA gene was confirmed by RT-PCR.


CONCLUSIONS
Construction of the recombinant plasmid was successful. The gfp gene and pacA gene in the plasmid was transcribed and expressed simultaneously in the transfected cells. Moreover, detection of GFP is simple, safe and effective for living cells.
Liu
Li
L
Department of Microbiology, West China Basic Medicine and Forensic College, Sichuan University, Chengdu 610041, China.
Jia
Wenxiang
W
Li
Xueru
X
D003731:Dental Caries / Q000517:prevention & control*; D004926:Escherichia coli / Q000235:genetics; D005453:Fluorescence; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005798:Genes, Bacterial; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D022541:Streptococcal Vaccines / Q000235:genetics* / Q000276:immunology; D013295:Streptococcus mutans / Q000235:genetics* / Q000276:immunology; D019444:Vaccines, DNA / Q000235:genetics* / Q000276:immunology
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D022541:Streptococcal Vaccines; D019444:Vaccines, DNA; D049452:Green Fluorescent Proteins