缺失TC7L-TK2L基因减毒天坛株痘苗病毒构建及筛选  

Construction and selection of the Tiantan strain of the vaccinia virus attenuated by deletion of the TC7L-TK2L gene

作　　者：李一权 阚式绂[2] 胡宁宁[2] 姜爽[2] 陈智飞[2] 周晔[2] 李霄[2] 孙丽丽[2,3] 金宁一[1,2] 

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]吉林省肿瘤医院头颈外一科,吉林长春130021

出　　处：《中国病原生物学杂志》2014年第11期961-964,974,共5页Journal of Pathogen Biology

基　　金：国家863计划项目(No.2012AA02A407);国家新药创制科技重大专项(No.2014ZX09304314-002);国家自然科学基金项目(No.81101140);吉林省产业技术创新战略联盟项目(No.20140309006YY);吉林省重点科技攻关项目(No.20130206041NY)

摘　　要：目的通过敲除天坛株痘苗病毒(Vaccinia virus Tiantan strain,VTT)的复制非必需基因TC7L-TK2L,并结合双标记筛选及外源筛选标记敲除技术构建TC7L-TK2L基因缺失的VTT弱毒株rVTT-TC7L--TK2L-。方法人工合成含有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)的重组臂,构建穿梭质粒pTC7L-TK2L-EGFP,再与VTT共转染BHK-21细胞,通过连续筛选10次,获得含有筛选标记的重组痘苗病毒rVTT-TC7L--TK2L--EGFP+;利用Cre/loxp系统敲除EGFP,连续筛选10次,获得无筛选标记的重组痘苗病毒rVTT-TC7L--TK2L-,用PCR方法对rVTT-TC7L--TK2L-进行鉴定和遗传稳定性检测,用电镜观察病毒形态变化,并通过绘制生长曲线观察病毒在细胞内的复制情况。结果 PCR结果表明,成功构建了重组痘苗病毒rVTT-TC7L--TK2L-,且具有良好的遗传稳定性;电镜观察痘苗病毒缺失TC7L-TK2L基因未影响病毒形态;生长曲线显示敲除TC7L-TK2L基因后病毒毒力显著下降,但不影响病毒的正常复制。结论成功构建了痘苗病毒减毒株rVTT-TC7L--TK2L-,该重组痘苗具有良好的遗传稳定性且毒力减弱。Objective To construct an attenuated Tiantan strain of the vaccinia virus（VTT）with deletion of the TC7LTK2 Lgene by combining double-label screening and deletion of an exogenous selection marker. Methods The shuttle plasmid pTC7L^-TK2L^-EGFP was constructed with arms engineered to contain an enhanced vaccinia virus early-late promoter（pE/L）and enhanced green fluorescent protein（EGFP）.BHK-21 cells were co-transfected with pTC7L^-TK2 LEGFP and VTT.In the recombinant virus rVTT-TC7L^--TK2L^-EGFP＋,the TC7L^-TK2 Lgene was replaced with the EGFP gene.This virus was screened with 10 rounds of fluorescent plaque purification.rVTT-TC7L^--TK2L^-with no exogenous selection marker was obtained by deleting selection markers with a Cre/Loxp system and another 10 rounds of fluorescent plaque purification.rVTT-TC7L^--TK2L^-was identified and its genetic stability was determined using PCR.The morphogenesis of rVTT-TC7L^--TK2L^-was observed using electron microscopy and viral replication was determined using agrowth curve. Results PCR results indicated that an attenuated strain of the vaccinia virus（rVTT-TC7L^--TK2L^-）was successfully constructed.Electron microscopy revealed that the virus＇ morphology was not affected by deletion of the TC7L^-TK2 Lgene.The growth curve revealed that the virus＇ virulence decreased markedly as a result of deletion of the TC7L^-TK2 Lgene but its replication was not affected. Conclusion An attenuated strain of the vaccinia virus（rVTT-TC7L^--TK2L^-）with good genetic stability was successfully constructed,and this strain had reduced virulenced.

关 键 词：重组天坛株痘苗病毒 病毒载体 同源重组 TC7L-TK2L基因 

分 类 号：R373.12[医药卫生—病原生物学]