T7噬菌体转运真核表达载体入胞表达平台的构建

Construction of T7 phage mediated eukaryotic expression vector transport platform

【目的】构建携带锚定序列的真核表达载体,研究T7噬菌体识别、包裹和转运真核表达载体进入细胞实现蛋白表达的可行性,为DNA疫苗研发建立新的技术平台。【方法】本研究通过重叠延伸PCR方法获得候选锚定序列并插入真核表达载体;建立荧光定量PCR方法比较T7噬菌体识别、包裹真核表达载体的效率;激光共聚焦显微镜观察T7噬菌体转运真核表达载体进入细胞实现报告基因的表达。【结果】获得4条锚定序列(AS1–4)并成功插入pcDNA3.0-EGFP真核表达载体;其中携带2号锚定序列(pcDNA3.0-EGFP-AS2)的真核表达载体可被T7噬菌体高效识别,对其包裹效率高达95%;T7噬菌体包裹真核表达载体可以抵御核酸酶对质粒的降解,并且转运真核表达载体进入树突状细胞内部实现报告基因EGFP的表达。【结论】该研究表明,T7噬菌体通过识别锚定序列将真核表达载体包裹进衣壳内部,完整的噬菌体颗粒作为转运工具将真核表达载体运送至细胞内部实现表达,这为DNA疫苗研发提供新的技术平台。

[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.