伪狂犬病毒5基因缺失株体外重组获得缺失基因的研究  

Homologous Recombination of 5 Gene-deletion Pseudorabies Virus with Wild Pseudorabies Virus in Cell Culture

作　　者：陈晓春[1] 韩爽 吴华伟[1] 邓永[1] 曹明慧[1] 李俊平[1] CHEN Xiao-chun;HAN Shuang;WU Hua-wei;DENG Yong;CAO Ming-hui;LI Jun-ping(China Institute of Veterinary Drug Control,Beijing 100081,China)

机构地区：[1]中国兽医药品监察所,北京100081 [2]青岛信得药业有限公司,山东青岛266100

出　　处：《中国兽药杂志》2018年第11期19-24,共6页Chinese Journal of Veterinary Drug

基　　金：国家重点研发计划项目"兽用生物制品质量检验和控制标准"(2017YFF0208600;2017YFF0208603)

摘　　要：为研究伪狂犬病毒缺失株体外重组情况,将伪狂犬病毒5基因缺失毒株PRV-gE-/gI-/US9-/△UL49.5/TK-株与野毒株等量混合,经PK15细胞连续传代至第10代,通过分析第10代培养物中毒株类型和比例了解缺失毒株体外同源重组的情况。结果发现,除PRV野毒株和PRV-gE-/gI-/US9-/△UL49.5/TK-株外,出现了5种新的基因组合类型,分别为PRV(gI-/gE-/US9-/TK-)、PRV(gI-/gE-/US9-/gN-)、PRV(gI-/gE-/US9-)、PRV(TK-)、PRV(gN-),病毒重组百分率为72%。重组毒株中以gI/gE/US9三基因缺失型为主,占重组蚀斑数的41.7%,占挑选蚀斑数的30%,高于野毒株和5基因缺失毒株的比例(26%和2%),成为主要的优势重组毒株。从各病毒类型比例上看,缺失株同时获得所有缺失基因的几率很小,且重组后的毒株趋向于稳定的自然缺失毒株(如gE自然缺失的Bartha株)。5种新的基因组合类型的毒株以103.0TCID50接种健康易感家兔,未出现伪狂犬病典型临床症状,安全性好。In order to study the recombination of Pseudorabies virus in vitro, the five gene-deletion strain PRVgE-/ gI- / US9- / △UL49.5/ TK and the PRV wild strain in equal amount were mixed. Then the viral mixture wascultured and passaged in PK15 for ten generations. To study the homologous recombination in vitro during theserial cell culture passage, the type and proportion of PRV strains within the tenbth generation culture wereanalyzed. Result of the analysis demonstrated five novel genotypes of recombinants emerged in addition to PRV-gE- / gI- / US9- / △UL49.5/ TK and wild strain. Those distinct recombinants included PRV（gE- / gI- / US9- / TK-）,PRV（gE- / gI- / US9- / gN-）, PRV（gE- / gI- / US9-）, PRV（TK-） and PRV（gN-）. Furthermore, the recombinantstrain with triple gene-deletion （gE/ gI/ US9） was found to be the major strain present, which percentage ishigher than wild type and original mutant by 26% and 2% respectively. This mutant also accounts for 41.7% of allrecombinant PFU and 30% of selected PFU, becoming the predominant strain in the population. Results show thatit is hard to get all missing gene fragments at the same time, and the recombinated strains are tend to stablenatural gene deleted mutant （e.g. Bartha, gE natural missing strains）. The five novel PRV recombinant strainswere safe to rabbit at 103.0 TCID50, which show that the safety is good.

关 键 词：伪狂犬病毒 基因缺失 同源重组 

分 类 号：S852.65[农业科学—基础兽医学]