基于CRISPR/Cas9介导的同源重组技术构建TK、gE和gI基因缺失的伪狂犬病病毒

Constructing pseudorabies viruses with TK,gE,and gI gene deletions based on CRISPR/Cas9-mediated homologous recombination techniques

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,对从湖北某猪场送检的病料中分离到的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、生长曲线测定及安全性和效力试验对基因缺失株的特性进行初步研究。结果显示: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株传代至30代,TK和gE/gI基因缺失部分序列稳定,不能恢复;PRV HB2017ΔTKΔgE/gI株对仔猪是安全的;将该基因缺失株以10^(6.0) TCID_(50)和10^(7.0) TCID_(50)的病毒剂量接种仔猪,可保护仔猪免受10^(8.0) TCID_(50)病毒剂量强毒的攻击。

In this study,CRISPR/cas9-mediated homologous recombination technology was used to genetically edit the pseudorabies virus(PRV)HB2017 strain isolated from a pig farm in Hubei Province,and a PRV with deletion of TK,gE and gI genes was constructed.Afterwards,the gene-deleted PRV strain was obtained by techniques such as plaque purification.Subsequently,characteristics of the attenuated PRV were preliminarily studied by PCR,gene sequencing,indirect immunofluorescence assay,growth curve determination,vaccine safety and efficacy tests.The results showed that the TK,gE and gI genes of the PRV HB2017 strain had 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 were not significantly different and had high virus titer.After the PRV HB2017ΔTKΔgE/gI strain was transmitted to the 30th generation,the deletion sequences of the TK and gE/gI genes were stable and could not be recovered.The PRV HB2017ΔTKΔgE/gI strain is safe for piglets.Vaccinating piglets with 10_(6.0 )TCID_(50) or 10^(7.0) TCID_(50) of the PRV HB2017ΔTKΔgE/gI strain could protect them from the attack of 10^(8.0) TCID_(50) of the PRV virulent strains.