Construction of T7 phage mediated eukaryotic expression vector transport platform

Abstract:

[Objective] This study aimed to construct a novel T7 phage delivery platform for the recognition and packaging of eukaryotic expression vector harboring an anchor sequence, and to evaluate its feasibility for DNA vaccine research and development.[Methods] Anchor sequences were prepared by SOE-PCR method and inserted into the non-essential region of pcDNA3.0-EGFP to construct the recombinant eukaryotic expression plasmid. The recognition and packaging efficiency of recombinant plasmids by T7 phage was determined via fluorescence quantitative PCR method. Intact T7 phage particles carrying recombinant plasmids were then used as vehicle to deliver plasmids into dendritic cells. The EGFP gene expression was detected using a confocal microscope.[Results] Four PCR amplified-anchor sequence (AS1-4) were successfully inserted into pcDNA3.0-EGFP plasmid. The recombinant plasmid pcDNA3.0-EGFP-AS2 could be recognized and packaged by T7 phage at a package efficiency of approximately 95%. T7 phage packaging effectively prevented the nuclease degradation of recombinant plasmids. Moreover, intense EGFP expression was detected by confocal microscopy suggesting the successful phage-based delivery of plasmids into dendritic cells.[Conclusion] Our results demonstrate that eukaryotic expression plasmid harboring anchor sequences can be recognized and packaged by T7 phage, and the intact phage particles can be used as a vehicle to delivery plasmids into dendritic cells for endogenous gene expression. T7 phage mediated eukaryotic expression may provide a novel technical platform for the research and development of DNA vaccine.