Establishment of a Fosmid Library for Pseudorabies Virus SC Strain and Application in Viral Neuronal Tracing
Establishment of a Fosmid Library for Pseudorabies Virus SC Strain and Application in Viral Neuronal Tracing
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伪狂犬病病毒SC株Fosmid文库的建立及其在病毒神经元示踪中的应用
DOI：
10.3389/fmicb.2020.01168
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发表时间：
2020-06-11
影响因子：
5.2
通讯作者：
Sun, Yuan
中科院分区：
文献类型：
--
作者：
Qi, Hansong;Wu, Hongxia;Sun, Yuan
伪狂犬病毒（Pseudorabies virus， PRV）是甲型疱疹病毒亚科的一员，其嗜神经性和潜伏性感染引起了许多科学家的关注。带有荧光报告基因标记的PRV作为跟踪器已被用于分析神经回路，包括逆行和逆行。在本研究中，我们利用fosmid文库构建了一个快速高效的重组PRV生成平台。首先，将高纯度PRV双城（SC）基因组DNA随机剪切为约30-49 kb的DNA片段。经末端钝化和磷酸化后，将DNA片段克隆到fosmid载体中，转化到大肠杆菌中。对覆盖PRV SC全基因组的200个化石片段进行了测序。将5组13个fosmid组合分别转染到Vero细胞中，每组都能成功拯救PRV株SC，野生型和重组型在形态和生长动力学上没有显著差异。下一步，利用Red/ET重组技术将增强型绿色荧光蛋白（EGFP）融合到UL36蛋白的氨基端，成功救出重组rPRV SC-UL36-EGFP。最后，在感染后0.5 ~ 2 h （hpi）监测到单个绿色荧光病毒颗粒在轴突内逆行运动的平均速度为0.71 +/- 0.43 μ m/s，在感染后8 hpi时监测到病毒颗粒在轴突内逆行运动的平均速度为0.75 +/- 0.49 μ m/s。结合fosmid文库和Red/ET重组技术构建PRV重组体高效、稳定。这项研究将加速对PRV生物学的认识和新型疫苗的开发。
Pseudorabies virus (PRV) is a member ofAlphaherpesvirinaesubfamily, 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 intoEscherichia coli. 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 mu m/s at 0.5-2 h post infection (hpi) and anterograde moving with an average velocity of 0.75 +/- 0.49 mu m/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.