A Novel and Simple Method for Rapid Generation of Recombinant Porcine Adenoviral Vectors for Transgene Expression

作者：Zhang, Peng; Du, Enqi; Ma, Jing; Wang, Wenbin; Zhang, Lu; Tikoo, Suresh K.^*; Yang, Zengqi

来源：PLos One, 2015, 10(5): e0127958.

DOI：10.1371/journal.pone.0127958

• CLASSICAL SWINE-FEVER
• GENE-TRANSFER
• NONHUMAN ADENOVIRUSES
• IMMUNE-RESPONSES
• INNATE IMMUNITY
• PROTECTS PIGS
• VACCINATION
• CONSTRUCTION
• EFFICIENT
• TOXICITY

Many human (different serotypes) and nonhuman adenovirus vectors are being used for gene delivery. However, the current system for isolating recombinant adenoviral vectors is either time-consuming or expensive, especially for the generation of recombinant nonhuman adenoviral vectors. We herein report a new and simple cloning approach for the rapid generation of a porcine adenovirus (PAdV-3) vector which shows promise for gene transfer to human cells and evasion of human adenovirus type 5 (HAdV-5) immunity. Based on the final cloning plasmid, pFPAV3-CcdB-Cm, and our modified SLiCE strategy (SLiCE cloning and lethal CcdB screening), the process for generating recombinant PAdV-3 plasmids required only one step in 3 days, with a cloning efficiency as high as 620 +/- 49.56 clones/ng and zero background (100% accuracy). The recombinant PAdV-3 plasmids could be successfully rescued in porcine retinal pigment epithelium cells (VR1BL), which constitutively express the HAdV-5 E1 and PAdV-3 E1B 55k genes, and the foreign genes were highly expressed at 24 h after transduction into swine testicle (ST) cells. In conclusion, this strategy for generating recombinant PAdV-3 vectors based on our modified SLiCE cloning system was rapid and cost-efficient, which could be used as universal cloning method for modification the other regions of PAdV-3 genome as well as other adenoviral genomes.

• 出版日期2015-5-26