tk1基因敲除Vero细胞株的建立及其在单纯疱疹病毒tk基因修复中的应用

Establishment of tk1-Knockout Vero Cell Line and Its Application in HSV tk Repair

利用单纯疱疹病毒I型(Herpes simplex virus type 1,HSV-1)标准株F细菌人工染色体(HSV-BAC)系统构建的HSV重组病毒由于存在tk基因缺陷,无法表达胸苷激酶,影响病毒潜伏/激活,故需对该基因进行修复。为此,本研究建立了tk1基因敲除的Vero细胞株(tk1-Vero),并利用该细胞株建立了次黄嘌呤-氨基蝶呤-胸腺嘧啶脱氧核苷(Hypoxanthine-aminopurine-thymidine,HAT)选择性培养液筛选方法,对经同源重组方式完成tk基因修复的病毒进行筛选。首先,利用CRISPR/Cas9基因编辑技术敲除Vero细胞tk1基因,通过有限稀释法建立tk1-Vero细胞株,采用DNA测序和Western Blot对tk1-Vero细胞株进行鉴定;比较HSV(F)和tk-HSV(F)在tk1-Vero细胞和tk基因缺陷的人骨肉瘤143细胞中的增殖情况;在tk1-Vero细胞中,采用HAT选择性培养液筛选方法,对tk基因修复病毒进行筛选。结果显示,本研究成功建立了稳定的tk1-Vero细胞株;与143细胞相比,tk1-Vero细胞更适于病毒增殖及tk基因修复病毒的筛选;最后在tk1-Vero细胞中,利用HAT选择性培养液成功筛选出tk基因修复病毒。本研究表明,采用tk1-Vero细胞株建立的HAT选择性培养液筛选方法可以对tk基因修复病毒进行高效快速筛选。

Construction of herpes simplex virus(HSV)mutants using bacterial artificial chromosome containing a copy of HSV-1(F)genome(HSV-BAC)usually results in thymidine kinase(tk)gene deficiency. Since thymidine kinase plays an important role in viral latency and reactivation,this gene in mutants must be rescued.We established a tk1-knockout Vero cell line and developed a procedure to select tk-repaired mutants by HAT medium screening in tk1-knockout Vero cell line. The tk1-knockout Vero cell line was established by CRISPR/Cas9 genome editing and limited dilution method,and then identified by DNA sequencing and Western Blot. In contrast to tk1-deficient 143 cells,tk1-knockout Vero cell line was more suitable for HSV replication and screening of tk-repaired mutants. By using tk1-knockout Vero cell line,HSV mutants in which tk had been rescued through homologous recombination were successfully selected by screening using HAT medium. These results demonstrated that selection of tk-repaired HSV mutants through HAT medium screening in tk1-knockout Vero cell line was effective and rapid.