College of Veterinary Medicine, North-west A&F University, Yangling, Shaanxi, China.
VIDO-InteVac, University of Saskatchewan Saskatoon, Saskatchewan, Canada; Vaccinology & Immunotherapeutics Program, School of Public Health, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
J Virol Methods. 2018 Nov;261:139-146. doi: 10.1016/j.jviromet.2018.08.020. Epub 2018 Aug 31.
Bovine adenovirus type 3 (BAdV3) has been widely used as a vector for vaccine research and development, whereas BAdV1 biology and BAdV1-based vectored vaccine have been less frequently reported. We aimed to construct an infectious BAdV1 clone and explore the functions of BAdV1 genes.
First, the infectious clone of pUCBAdV1 containing the full-length BAdV1 DNA and the recombinant plasmid pUCBAV1-EYFP expressing the marker gene EYFP were constructed. Then, the recombinant viruses BAdV101 and rBAdV1-EYFP were rescued. The stability of the exogenous EYFP gene was analyzed by continuous passage, PCR, and western blotting. Finally, the virus neutralization titer of the rescued viruses was evaluated.
The infectious clones of pUCBAdV1 and pUCBAV1-EYFP were constructed and the recombinant viruses BAdV101 and rBAdV1-EYFP were rescued successfully. Moreover, the results showed that the EYFP gene could be expressed continuously. In addition, the replication of rBAdV1-EYFP was less efficient than that of the wild-type virus wtBAdV1 in vitro, while the efficacy of BAdV101 replication was almost the same as that of wtBAdV1. Furthermore, the neutralization test showed that the neutralization titer of rBAdV1-EYFP was consistent with that of wtBAdV1.
To our knowledge, the infectious genome of pUCBAV1-EYFP expressing a visible marker gene EYFP was constructed for the first time, and the finding forms a basis for the development of BAdV1-based efficient vectored vaccine.
牛腺病毒 3 型（BAdV3）已广泛用作疫苗研发的载体，而 BAdV1 的生物学特性及其基于 BAdV1 的载体疫苗的研究则相对较少。本研究旨在构建传染性 BAdV1 克隆并探索 BAdV1 基因的功能。
首先，构建了含有全长 BAdV1 DNA 的 pUCBAdV1 感染性克隆和表达标记基因 EYFP 的重组质粒 pUCBAV1-EYFP。然后，拯救出重组病毒 BAdV101 和 rBAdV1-EYFP。通过连续传代、PCR 和 Western blot 分析来检测外源 EYFP 基因的稳定性。最后，评估了拯救病毒的病毒中和效价。
成功构建了 pUCBAdV1 和 pUCBAV1-EYFP 感染性克隆，并拯救出重组病毒 BAdV101 和 rBAdV1-EYFP。结果表明，EYFP 基因可以连续表达。此外，rBAdV1-EYFP 的复制效率在体外比野生型病毒 wtBAdV1 低，而 BAdV101 的复制效率几乎与 wtBAdV1 相同。此外，中和试验表明，rBAdV1-EYFP 的中和效价与 wtBAdV1 一致。
据我们所知，首次构建了表达可见标记基因 EYFP 的 pUCBAV1-EYFP 感染性基因组，为开发基于 BAdV1 的高效载体疫苗奠定了基础。
