中国动物传染病学报
中國動物傳染病學報
중국동물전염병학보
CHINESE JOURNAL OF VETERINARY PARASITOLOGY
2011年
6期
25-30
,共6页
刘振江%刘昊%刘燕瑜%郭欢欢%凡敏%岳云强%陆飞%秦艳青%李国江%鲁会军%金宁一
劉振江%劉昊%劉燕瑜%郭歡歡%凡敏%嶽雲彊%陸飛%秦豔青%李國江%魯會軍%金寧一
류진강%류호%류연유%곽환환%범민%악운강%륙비%진염청%리국강%로회군%금저일
辛德毕斯病毒%转移载体%绿色荧光蛋白%重组痘苗病毒
辛德畢斯病毒%轉移載體%綠色熒光蛋白%重組痘苗病毒
신덕필사병독%전이재체%록색형광단백%중조두묘병독
Sindbis virus%transfer vector%enhanced green fluorescent protein （EGFP）%recombinant vaccinia virus
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。
為瞭構建錶達辛德畢斯病毒E基因的重組痘苗病毒,本研究通過基因重組的方法將辛德畢斯病毒E基因及綠色熒光蛋白（enhanced green fluorescent protein,EGFP）基因分彆連接至痘苗病毒轉移載體pSTK,酶切鑒定得到暘性重組質粒pSTK-SINE-EGFP。採用脂質體轉染的方法,將該重組質粒與痘苗病毒天罈株共轉染BHK-21細胞,通過同源重組穫得重組痘苗病毒。利用EGFP篩選暘性重組痘苗病毒vTTVV-SINE-EGFP,收集感染重組痘苗病毒的BHK-21細胞,SDS-PAGE電泳檢測辛德畢斯病毒E基因在細胞中的錶達情況,Western blot分析錶達產物的免疫原性。結果證明辛德畢斯病毒E基因能在重組痘苗病毒vTTVV-SINE-EGFP中穫得錶達,且錶達產物具有良好的免疫原性。錶達辛德畢斯病毒E基因的重組痘苗病毒的成功構建,為研製辛德畢斯病毒活載體疫苗奠定瞭基礎。
위료구건표체신덕필사병독E기인적중조두묘병독,본연구통과기인중조적방법장신덕필사병독E기인급록색형광단백（enhanced green fluorescent protein,EGFP）기인분별련접지두묘병독전이재체pSTK,매절감정득도양성중조질립pSTK-SINE-EGFP。채용지질체전염적방법,장해중조질립여두묘병독천단주공전염BHK-21세포,통과동원중조획득중조두묘병독。이용EGFP사선양성중조두묘병독vTTVV-SINE-EGFP,수집감염중조두묘병독적BHK-21세포,SDS-PAGE전영검측신덕필사병독E기인재세포중적표체정황,Western blot분석표체산물적면역원성。결과증명신덕필사병독E기인능재중조두묘병독vTTVV-SINE-EGFP중획득표체,차표체산물구유량호적면역원성。표체신덕필사병독E기인적중조두묘병독적성공구건,위연제신덕필사병독활재체역묘전정료기출。
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.
