天坛株痘苗病毒基因缺失弱毒株的构建及鉴定  

Construction and identification of the attenuated vaccinia Tiantan strain deleted for multiple genes

作　　者：盛媛[1,2] 王浩然[3] 胡宁宁[2] 陈智飞[2,3] 王宏宇[1,2] 姜爽[2] 周晔[2] 李一权 李霄[2] 金宁一[2] 

机构地区：[1]吉林农业大学动物科学技术学院,吉林长春130118 [2]军事医学科学院军事兽医研究所,吉林长春130122 [3]吉林大学农学部,吉林长春130062

出　　处：《中国兽医学报》2014年第3期384-389,共6页Chinese Journal of Veterinary Science

基　　金：吉林省重点科技攻关资助项目(20130206041NY)

摘　　要：本研究旨在通过敲除部分与天坛株痘苗病毒(vaccinia virus Tiantan strain,VTT)毒力和宿主范围等相关的复制非必需基因,并结合双标记筛选及外源筛选标记敲除技术,构建多基因缺失VTT弱毒株。人工合成7对重组臂,结合痘病毒早晚期复合强启动子pE/L和外源筛选标记增强型绿色荧光蛋白(Enhance green fluorescent protein,EGFP),构建穿梭载体质粒pTC-EGFP、pTA35-EGFP、pTA66-EGFP、pTE-EGFP、pTB-EGFP、pTI-EGFP和pTJ-EGFP。将上述质粒依次与VTT或基因缺失VTT共转/感染BHK细胞,并通过同源重组和荧光蚀斑筛选方法,逐一敲除拟缺失基因片段(TC:TC7L^TK2L;TA35:TA35L;TA66:TA66R;TE:TE3L;TB:TB13R^TB14R;TI:TI4L;TJ:TJ2R)。利用Cre/Loxp系统实现对外源筛选标记的敲除,最终获得天坛株痘苗病毒基因缺失弱毒株TTVAC7,并运用PCR方法对TTVAC7进行鉴定和遗传稳定性检测。结果表明,本研究成功构建了缺失7个目的片段的天坛株痘苗病毒弱毒株TTVAC7,且该弱毒株具有良好的遗传稳定性。The research was designed to build an attenuated strain of vaccinia virus Tiantan strain （VTT）, by knocking out some ORFs of the genome of VTT non-essential for virus replication which were related to virulence and host range and so on, combined with double-label screening and exogenous selection marker knock out technology. In this study, seven shuttle vector plasmids were built named as pTC-EGFP, pTA35-EGFP, pTA66-EGFP, pTE-EGFP, pTB-EGFP, pTI-EG- FP and pTJ-EGFP, including seven pairs synthetic recombinant arms of target genes, as well as poxvirus sooner or later compound strong promoter pE/L and exogenous selection marker EGFP. These plasmids above were co-transfected/infeeted BHK cells with VTT or gene-deleted VTT one by one, integrated with homologous recombination and fluorescence plaque screening method, in order to knock out gene fragments which were proposed deleted as follows TC：TC7L-TK2L; TA35：TA35L; TA66： TA66R; TE： TE3L; TB： TB13R-TB14R; TI： TI4L; TJ： TJ2R. Using the Cre/Loxp system to knock out exogenous selection marker,we obtained the recombinant attenua- ted virus named as TTVAC7, then identified and detected its genetic stability by application of PCR method. The results showed that an attenuated strain of VTT TTVAC7 was successfully constructed,and the attenuated strain has a good genetic stability.

关 键 词：天坛株痘苗病毒 基因缺失 同源重组 增强型绿色荧光蛋白 

分 类 号：S852.65[农业科学—基础兽医学]