缺失E3L基因减毒天坛株痘苗病毒的构建及筛选  

Construction and selection of the attenued vaccinia Tiantan strain with deleted E3L gene

作　　者：王宇航[1,2,3] 李霄[2,3] 王浩然[1,2] 阚式绂[1,2,3] 王卓越[1,2,3] 齐延新[1,2,3] 吴娜[2,3] 杜寿文[1,2,3] 金宁一[2,3] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062 [2]军事医学科学院军事兽医研究所,吉林长春130062 [3]吉林省人兽共患病预防与控制重点实验室,吉林长春130062

出　　处：《中国兽医学报》2011年第11期1619-1622,共4页Chinese Journal of Veterinary Science

基　　金：转基因生物新品种培育重大专项(2009ZX08006-002B);"重大新药创制"科技重大专项(2010ZX09401-305-14);吉林省重大科技攻关项目(10ZDGG007)

摘　　要：通过在E3L同源重组臂中插入含有增强型痘苗病毒早晚期启动子(pE/L)、增强型绿色荧光蛋白基因和同向Loxp(Locus of X-over P1)序列的表达盒构建穿梭质粒pTE-EGFP。利用pTE-EGFP和野生型天坛株痘苗病毒共转染仓鼠肾细胞(BHK-21),通过对绿色荧光蚀斑的10次筛选,构建缺失E3L基因并含有EGFP基因的重组痘苗病毒rTTVV-TE--EGFP+。利用rTTVV-TE--EGFP+和含有Cre(Cyclization recombination)重组酶基因的重组质粒pVAX1-Cre共转染BHK-21细胞,通过对无荧光蚀斑的10次筛选,构建无筛选标记的天坛株痘苗病毒减毒株rTTVV-TE--,并利用聚合酶链式反应(PCR)对其进行鉴定。鉴定结果显示,rTTVV-TE--中无E3L基因转录和EGFP基因表达。以上结果说明,所构建的痘苗病毒减毒株rTTVV-TE--完全缺失了E3L基因和外源筛选标记EGFP基因,且具有良好的遗传稳定性。To construct E3＋. deleted Tiantan vaccinia virus,a shuttle plasmid pTE-EGFP was generated which in addition to the E3L homologous recombinant arms contained an enhanced vaccinia virus early-late promoter, pE/L, fol- lowed by enhanced green fluorescent protein （EGFP） gene flanked by two syntropic locus of X-over P1 （Loxp） se- quences. Baby hamster kidney cells （BHK-21） cells were then co-transfected with pTE-EGFP and the wild vaccinia Tiantan strain. The modified vaccinia virus rTTVV-TE -EGFP＋ in which E3L gene was replaced with the EGFP gene was screened by 10 rounds of fluorescent plaque purification. To knock out the selection marker EGFP gene, rTTVV-TE -EGFP＋ and the plasmid pVAX I -Cre containing Cre gene were co-transfected into BHK-21 cells. The attenued vaccinia Tiantan strain rTTVV-TE-- without {orign screen signal was selected by another 10 rounds of non-fluorescent plaque prufication and identified by polymerase chain reaction （PCR）. The results showed that neither transcript of the E3L gene nor expression of the EGFP gene was detected,indicating that the E3L gene and the EGFP gene had been removed completely form rTTVV-TE -,and the attenued vaceinia Tiantan strain was geneti- cally stable.

关 键 词：天坛株痘苗病毒 减毒病毒 E31. Cre-Loxp系统 病毒载体 

分 类 号：S852.65[农业科学—基础兽医学]