摘要
通过敲除痘苗病毒天坛株(vaccinia virus tiantan strain,VTT)复制非必需基因TA35R,结合双筛选标记及同源重组技术,构建TA35R基因缺失的VTT弱毒株rVTT-TA35R-。人工合成含有TA35R重组臂、同向loxp序列、早晚期启动子PE/L、EGFP及酶切位点的穿梭质粒pTA35R-EGFP。pTA35R-EGFP和野生型VTT共转染BHK-21细胞,通过绿色荧光蚀斑筛选,构建缺失TA35R基因且含FGFP基因的重组痘苗病毒rVTT-TA35R--EGFP+。采用Cre/Loxp系统敲除外源筛选标记EGFP,获得缺失TA35R基因的重组痘苗病毒rVTT-TA35R-,利用PCR方法和电子显微镜对其进行鉴定,通过MTT法检测细胞噬性以评价其减毒效果。结果表明,所构建的痘苗病毒弱毒株rVTT-TA35R-完全缺失了TA35R基因和外源筛选标记EGFP,且细胞毒性减弱。
The attenuated vaccinia Tiantan strain(VTT)with deleted TA35 R gene(rVTTTA35R-)was constructed through the technology of homologous recombination,double marking screening and gene knockout.Shuttle vector plasmids named as pTA35R-EGFP was compound of synthetic recombinant arms of TA35 R,loxp sequence in the same direction,EGFP,sooner or later promoter PE/L and restriction enzyme cutting site.BHK-21 cells were co-transfected with pTA35R-EGFP and wild type VTT.The modified virus rVTT-TA35R--EGFP+in which TA35 Rgene was replaced with the EGFP gene was screened by fluorescent plaque purification.rVTT-TA35R-without exogenous screen signal was obtained by knocking out selection markers with Cre/Loxp system and identified by PCR and electron microscope,and attenuated capability evaluation by MTT assay.The results showed that the TA35 Rgene and the EGFP gene had been removed completely from rVTT-TA35R-,and the cell virulence was declined.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2014年第12期1883-1887,共5页
Chinese Journal of Veterinary Science
基金
国家863计划资助项目(2012AA02A407)
国家新药创制科技重大专项资助项目(2014Z×09304314-002)
国家自然科学基金资助项目(81101140)
吉林省产业技术创新战略联盟资助项目(20140309006YY)
吉林省重点科技攻关资助项目(2013206041NY)
