Qi Hansong, Wu Hongxia, Abid Muhammad, Qiu Hua-Ji, Sun Yuan
State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Front Microbiol. 2020 Jun 11;11:1168. doi: 10.3389/fmicb.2020.01168. eCollection 2020.
Pseudorabies virus (PRV) is a member of subfamily, its neurotropism and latency infection attract the attention of many scientists. PRV tagged with a fluorescent reporter gene as a tracker has been used to analyze neuronal circuits, including anterograde and retrograde. In this study, we used fosmid library to construct a rapid and efficient platform to generate recombinant PRV. Firstly, the highly purified PRV ShuangCheng (SC) genomic DNA was sheared randomly into approximately 30-49-kb DNA fragments. After end-blunting and phosphorylation, the DNA fragments were cloned into the fosmid vector and transformed into . A total of 200 fosmids that cover the complete genome of PRV SC was sequenced. Thirteen fosmid combinations in five groups were transfected into Vero cells, respectively, and each group can successfully rescue PRV strain SC. There was no significant difference between wild type and recombinant in both morphology and growth kinetics. In the next step, an enhanced green fluorescent protein (EGFP) was fused into the amino-terminal of UL36 protein by Red/ET recombination technology, and recombinant rPRV SC-UL36-EGFP was rescued successfully. At last, the single viral particles with green fluorescent were monitored retrograde moving in the axon with an average velocity of 0.71 ± 0.43 μm/s at 0.5-2 h post infection (hpi) and anterograde moving with an average velocity of 0.75 ± 0.49 μm/s at eight hpi. Integration of fosmid library and Red/ET recombination technology in our work was highly efficient and stable for constructing PRV recombinants. This study will accelerate understanding the biology of PRV and the development of novel vaccines.
伪狂犬病病毒（PRV）是亚科的成员，其嗜神经性和潜伏感染吸引了许多科学家的关注。带有荧光报告基因作为追踪器的PRV已被用于分析神经回路，包括顺行和逆行神经回路。在本研究中，我们使用fosmid文库构建了一个快速高效的平台来产生重组PRV。首先，将高度纯化的PRV双城（SC）基因组DNA随机剪切成约30 - 49 kb的DNA片段。经末端平端化和磷酸化后，将DNA片段克隆到fosmid载体中并转化到……。总共对覆盖PRV SC完整基因组的200个fosmid进行了测序。将五组中的13种fosmid组合分别转染到Vero细胞中，每组均能成功拯救PRV毒株SC。野生型和重组体在形态和生长动力学方面均无显著差异。下一步，通过Red/ET重组技术将增强型绿色荧光蛋白（EGFP）融合到UL36蛋白的氨基末端，成功拯救了重组rPRV SC-UL36-EGFP。最后，监测到感染后0.5 - 2小时（hpi）绿色荧光单病毒颗粒在轴突中逆行移动，平均速度为0.71±0.43μm/s，感染后8小时顺行移动，平均速度为0.75±0.49μm/s。在我们的工作中，fosmid文库和Red/ET重组技术的整合对于构建PRV重组体高效且稳定。本研究将加速对PRV生物学的理解以及新型疫苗的开发。
