重组辛德毕斯病毒E基因痘苗病毒的构建  

CONSTRUCTION OF RECOMBINANT VACCINIA VIRUS EXPRESSING E GENE OF SINDBIS VIRUS

作　　者：刘振江[1,2] 刘昊[1,3] 刘燕瑜[1,3] 郭欢欢[1,2] 凡敏[1,2] 岳云强[1,2] 陆飞[1,2] 秦艳青[1,2] 李国江[2,4] 鲁会军[1] 金宁一[1] 

机构地区：[1]中国人民解放军军事医学科学院军事兽医研究所,长春130122 [2]吉林农业大学动物科技学院,长春130118 [3]吉林大学畜牧兽医学院,长春130062 [4]吉林农业科技学院,吉林132101

出　　处：《中国动物传染病学报》2011年第6期25-30,共6页Chinese Journal of Animal Infectious Diseases

基　　金：公益性行业科研专项(201203082)

摘　　要：为了构建表达辛德毕斯病毒E基因的重组痘苗病毒,本研究通过基因重组的方法将辛德毕斯病毒E基因及绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因分别连接至痘苗病毒转移载体pSTK,酶切鉴定得到阳性重组质粒pSTK-SINE-EGFP。采用脂质体转染的方法,将该重组质粒与痘苗病毒天坛株共转染BHK-21细胞,通过同源重组获得重组痘苗病毒。利用EGFP筛选阳性重组痘苗病毒vTTVV-SINE-EGFP,收集感染重组痘苗病毒的BHK-21细胞,SDS-PAGE电泳检测辛德毕斯病毒E基因在细胞中的表达情况,Western blot分析表达产物的免疫原性。结果证明辛德毕斯病毒E基因能在重组痘苗病毒vTTVV-SINE-EGFP中获得表达,且表达产物具有良好的免疫原性。表达辛德毕斯病毒E基因的重组痘苗病毒的成功构建,为研制辛德毕斯病毒活载体疫苗奠定了基础。To construct recombinant vaccinia virus expressing E gene of Sindbis virus, Sindbis virus E gene and green fluorescent protein （EGFP） gene were inserted to the vaccinia virus transfer vector pSTK by gene recombination. The resulting recombinant plasmid pSTK-SINE-EGFP was confirmed in restriction enzyme digestion. Subsequently, the recombinant plasmid and vaccinia virus Tiantan strain were co-transfected into BHK-21 cells and positive recombinant vaccinia virus was identified by fluorescent plaque purification. Sindbis virus E gene was expressed in BHK-21 cells infected with the recombinant vaccinia virus as determined in SDS-PAGE and Western blot. The results demonstrated that recombinant vaccinia virus expressing Sindbis virus E gene was constructed and expressed product showed good immunogenicity, which laid a solid foundation for development of a live vector vaccine of Sindbis virus.

关 键 词：辛德毕斯病毒 转移载体 绿色荧光蛋白 重组痘苗病毒 

分 类 号：S852.659.6[农业科学—基础兽医学] Q78[农业科学—兽医学]