基于猪圆环病毒2型复制子的复制型表达载体的构建

Construction of a replicative expression vector based on the porcine circovirus 2 replicon

摘要:

DNA疫苗在动物体内表达抗原基因的水平是决定DNA疫苗免疫效果的关键，而提高DNA疫苗抗原基因表达水平的途径之一是利用可在动物细胞内复制的质粒作为载体。本研究利用pcDNA3.1构建了1个基于猪圆环病毒2（porcine circovirus 2，PCV2）复制子的复制型DNA疫苗载体pCMVori，再将增强型绿色荧光蛋白（enhanced green fluorescent protein，EGFP）基因分别克隆入pCMVori和对照质粒pcDNA3.1中，然后分别转染PK-15细胞，48 h后观察两组转染细胞EGFP表达情况后分别提取细胞质粒和RNA。利用Bcl I酶切前、后的质粒为模板通过定量PCR检测质粒的复制效率，并通过反转录聚合酶链式反应（reverse transcription polymerase chain reaction，RT-PCR）检测PCV2复制子的Rep基因转录产物。通过Image J软件分析两组转染细胞的平均荧光强度，并通过定量RT-PCR定量检测两组转染细胞的EGFP基因转录水平。结果显示，pCMVori在细胞内48 h的复制效率达到136%，RT-PCR结果证实Rep和Rep’均有转录。pCMVori-EGFP转染细胞的平均荧光强度比pcDNA3.1-EGFP的高39.14%，定量RT-PCR检测结果显示，前者的EGFP转录水平也比后者高40%。本研究结果表明，所构建的复制型DNA疫苗载体pCMVori可在真核细胞中独立复制，克隆的目的基因的表达水平也因此获得提升，为构建复制型DNA疫苗奠定了基础。

关键词:DNA复制子;表达载体;猪圆环病毒2型;DNA疫苗

Abstract:

The antigen gene expression level of a DNA vaccine is the key factor influencing the efficacy of the DNA vaccine. Accordingly, one of the ways to improve the antigen gene expression level of a DNA vaccine is to utilize a plasmid vector that is replicable in eukaryotic cells. A replicative DNA vaccine vector pCMVori was constructed based on the non-replicative pcDNA3.1 and the replicon of porcine circovirus 2 (PCV2) in this study. An EGFP gene was cloned into pCMVori and the control plasmid pcDNA3.1. The two recombinant vectors were transfected into PK-15 cell, and the plasmid DNA and RNA were extracted from the transfected cells. Real-time PCR was used to determine the plasmid replication efficiency of the two plasmids using plasmid before and after Bcl I digestion as templates, and the transcription level of the Rep gene in PCV2 replicon was detected by RT-PCR. The average fluorescence intensity of cells transfected with the two plasmids was analyzed with software Image J, and the transcription level of EGFP was determined by means of real-time RT-PCR. The results showed that the replication efficiency of pCMVori in PK-15 cells incubated for 48 h was 136%, and the transcriptions of Rep and Rep' were verified by RT-PCR. The average fluorescence intensity of the cells transfected with pCMVori-EGFP was 39.14% higher than that of pcDNA3.1-EGFP, and the transcription level of EGFP in the former was also 40% higher than that in the latter. In conclusion, the DNA vaccine vector pCMVori constructed in this study can independently replicate in eukaryotic cells. As a result, the expression level of cloned target gene was elevated, providing a basis for developing the pCMVori-based DNA vaccine.

Key words:DNA replicon;expression vector;porcine circovirus 2;DNA vaccine