A novel and simple method for rapid generation of recombinant porcine adenoviral vectors for transgene expression.

Peng Zhang,Jing Ma,Enqi Du,Zengqi Yang,Lu Zhang,Wenbin Wang,Suresh K Tikoo,Baochuan Lin

doi:10.1371/journal.pone.0127958

Abstract

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 non-human 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.

Highlights

Adenovirus-based vectors have been demonstrated to be excellent gene delivery vehicles in mammals [1,2,3,4], and certain features of these vectors have made them potential candidates for vaccine development and gene therapy [5]
The absence of cross-neutralization by sera containing Human adenovirus serotype 5 (HAdV-5)-specific antibodies and circumventing the preexisting immunity in humans have helped to initiate the evaluation of porcine adenovirus serotype 3 (PAdV-3) as a vector for the delivery of vaccine antigens in humans [12]
PAdV-3 is being developed as a vaccine delivery vehicle, the efficient evaluation of the PAdV-3 vector system requires the rapid generation of PAdV-3 vectors

Summary

Introduction

Adenovirus-based vectors have been demonstrated to be excellent gene delivery vehicles in mammals [1,2,3,4], and certain features of these vectors have made them potential candidates for vaccine development and gene therapy [5]. Rapid Generation of Porcine Adenovirus Vectors and the transgene can be expressed at a high level by strong heterologous promoters. These vectors can be grown to high titers in tissue culture (109–1010 infectious units per ml, IFU/ml). The absence of cross-neutralization by sera containing HAdV-5-specific antibodies and circumventing the preexisting immunity in humans have helped to initiate the evaluation of porcine adenovirus serotype 3 (PAdV-3) as a vector for the delivery of vaccine antigens in humans [12]. PAdV-3 is being developed as a vaccine delivery vehicle, the efficient evaluation of the PAdV-3 vector system requires the rapid generation of PAdV-3 vectors

Methods

Results

Conclusion