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[Construction of two eukaryotic expression plasmids pcDNA3/pacA and pcDNA3/pacP expressing surface protein antigen of
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.
Conclusion:
These two plasmids may act as gene vaccines for further study.