Establishment of a Fosmid Library for Pseudorabies Virus SC Strain and Application in Viral Neuronal Tracing

文献类型： 外文期刊

第一作者： Qi, Hansong

作者： Qi, Hansong;Wu, Hongxia;Abid, Muhammad;Qiu, Hua-Ji;Sun, Yuan

作者机构：

关键词： pseudorabies virus; fosmid library; UL36; neuron; transport

期刊名称：FRONTIERS IN MICROBIOLOGY （ 影响因子：5.64； 五年影响因子：6.32 ）

ISSN： 1664-302X

年卷期： 2020 年 11 卷

页码：

收录情况： SCI

摘要： 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.

分类号：

  • 相关文献
作者其他论文 更多>>

微信小程序