Construction of a replicative expression vector based on the porcine circovirus 2 replicon.
- Author:
Xiaoxue CAI
1
;
Jun LI
1
;
Zhangxun LI
1
;
Hongxu DU
1
;
Liting CAO
1
;
Yue MA
1
Author Information
- Publication Type:Journal Article
- Keywords: DNA replicon; DNA vaccine; expression vector; porcine circovirus 2
- MeSH: Animals; Swine; Circovirus/genetics*; Vaccines, DNA/genetics*; Replicon/genetics*; Genetic Vectors/genetics*; Plasmids/genetics*
- From: Chinese Journal of Biotechnology 2023;39(7):2634-2643
- CountryChina
- Language:Chinese
- 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.
