[Construction of two eukaryotic expression plasmids pcDNA3/pacA and pcDNA3/pacP expressing surface protein antigen of Streptococcus mutans].
OBJECTIVE
Two gene vaccines which encoded amino terminal and intermediate region in the PAc protein were constructed and the feasibility and safety was investigated, in order to obtain a highly effective and secure anticarious way by immunization.


METHODS
The target gene fragments pac-A and pac-P encoded amino terminal and intermediate region of surface protein antigen from Streptococcus mutans were obtained by PCR amplification respectively. Amplification products were isolated by using low melting-temperature agarose and recoveried from the gel. Then the two fragments were digested by Kpn I and Xho I, and ligated to the Kpn I-Xho I-digested eukaryotic shuttle expression vector pcDNA3 to create reconstructed plasmids pcDNA3/pacA and pcDNA3/pacP respectively. The ligated DNAs were transformed into Escherichia coli XL1-Blue. Transformed colonies carrying eukaryotic expression plasmid either pcDNA3/pacA or pcDNA3/pacP were screened by colony hybridization in situ. Then the reconstructed plasmids were isolated and identified by restricted endonuclease digestions, Southern hybridization and DNA sequencing.


RESULTS
1. Two DNA fragments pac-A and pac-P of the predicted size 1.3 kb and 1.2 kb were obtained by PCR amplified. 2. Eukaryotic expression plasmids pcDNA3/pacA and pcDNA3/pacP had been reconstructed correctly. 3. The sequencing data also indicated that the pcDNA3 construct contained the correct sequence and orientation of the two target genes.


CONCLUSIONS
These two plasmids may act as gene vaccines for further study.
Guo
L
L
College of Stomatology, West China University of Medical Sciences.
Liu
T
T
Chen
Z
Z
D000954:Antigens, Surface / Q000096:biosynthesis / Q000235:genetics*; D018985:Epitopes, B-Lymphocyte / Q000235:genetics; D018984:Epitopes, T-Lymphocyte / Q000235:genetics; D005057:Eukaryotic Cells / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D013295:Streptococcus mutans / Q000276:immunology*; D019444:Vaccines, DNA / Q000235:genetics
D000954:Antigens, Surface; D018985:Epitopes, B-Lymphocyte; D018984:Epitopes, T-Lymphocyte; D019444:Vaccines, DNA