Wuhan Science and Technology Plan Project
为构建TK、gE和gI基因缺失的伪狂犬病病毒（pseudorabies virus, PRV)，采用CRISPR/Cas9介导的同源重组技术对实验室分离的PRV HB2017株进行基因编辑，并利用蚀斑纯化等方法获取基因缺失株。随后，采用PCR、基因测序、间接免疫荧光试验、生长曲线测定及动物实验等对基因缺失株的特性进行了初步研究。结果表明：PRV HB2017株的TK、gE和gI基因已缺失，缺失毒株PRV HB2017ΔTKΔgE/gI与亲本毒株PRV HB2017株在PK-15细胞中的生长曲线差异不明显且具有较高的病毒滴度，PRV HB2017ΔTKΔgE/gI株具有安全性高、遗传稳定性和免疫原性好等优点。
In this study, CRISPR/Cas9-mediated homologous recombination technology was used to perform gene editing on the isolated PRV HB2017 strain to construct a pseudorabies virus (PRV) with deletion of TK, gE and gI genes. After techniques such as plaque purification, we have obtained the gene-deleted PRV strain. Subsequently, the characteristics of attenuated PRV were preliminarily studied by PCR, gene sequencing, indirect immunofluorescence assay, growth curve determination and animal experiments. The results showed that the TK, gE and gI genes of the PRV HB2017 strain have been deleted, and the growth curves of the attenuated PRV strain PRV HB2017ΔTKΔgE/gI and the parent strain PRV HB2017 strain in PK-15 cells are not significantly different and have high virus titer. PRV HB2017ΔTKΔgE/gI strain has the advantages of high safety, genetic stability and superior immunogenicity.