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
DOI:
作者:
作者单位:

作者简介:

通讯作者:

中图分类号:

基金项目:

武汉市科技计划项目(2019020702011378); 武汉东湖高新区‘3551光谷人才计划’; 湖北省技术创新专项重大项目(2017ABA056)


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

Fund Project:

  • 摘要
  • |
  • 图/表
  • |
  • 访问统计
  • |
  • 参考文献
  • |
  • 相似文献
  • |
  • 引证文献
  • |
  • 资源附件
  • |
  • 文章评论
    摘要:

    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106.0 TCID50和107.0 TCID50的病毒剂量接种仔猪,可保护仔猪免受108.0TCID50病毒剂量强毒的攻击。

    Abstract:

    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,gEand 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 106.0 TCID50 or 107.0 TCID50 of the PRV HB2017ΔTKΔgE/gI strain could protect them from the attack of 108.0 TCID50 of the PRV virulent strains.

    参考文献
    相似文献
    引证文献
引用本文

张华伟,周明光,侯真真,朱娴静,郝根喜,金建云,徐高原.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[J].华中农业大学学报,2021,40(2):206-212

复制
分享
文章指标
  • 点击次数:
  • 下载次数:
  • HTML阅读次数:
  • 引用次数:
历史
  • 收稿日期:2020-05-11
  • 最后修改日期:
  • 录用日期:
  • 在线发布日期: 2021-04-16
  • 出版日期: