摘要
将源于马立克氏病病毒 ( MDV) GA株的 Meq基因克隆到杆状病毒转移载体质粒 p Blu Bac4中强有力的多角化蛋白启动子 ( PPH)之后 ,经与线性化的 Bac-N-Blue TMDNA共转染于昆虫细胞系 SF9,获得了重组杆状病毒。使用重组痘病毒表达的 Meq制备的单抗 ,对重组杆状病毒感染的 SF9细胞及其裂解物分别进行间接免疫荧光试验、Western blotting和免疫沉淀试验 ,结果发现 :( 1 )本表达系统产生的 Meq可被原制备的单抗所识别 ;( 2 )在感染细胞中 ,Meq蛋白仅局限于细胞核内 ,而且随着感染后时间的增加 ,具有从核质向核仁和核膜转移的趋向 ;( 3 ) Western blotting和免疫沉淀试验均证实重组杆状病毒感染细胞裂解物中出现有 2条特异带 ( 60 0 0 0处 )。结果表明 ,杆状病毒
A recombinant baculovirus transfer vector pBluBac4Meq was constructed by cloning Meq gene of Marek′s Disease Virus(MDV) GA strain into the baculovirus transfer vector pBluBac4 under the polyhedrin promoter. The recombinant Meq baculovirus was obtained by co transfecting the insect SF9 cells with pBluBac4Meq and linearised Bac N Blue TM DNA. The recombinant baculovirus was selected by plaque assay and confirmed by PCR technique and sequencing of the inserted gene. The protein product of Meq gene was shown to be highly expressed in the nuclei of recombinant baculovirus infected SF9 cells when using an anti Meq monoclonal antibody(McAb) to run the immunofluorescence(IF) test; The expression quantity and IF staining patterns differed with times of postinfection. The results of Western blotting and immunoprecipitation test showed there were two specific bands around 60 000. The results demonstrated that the baculovirus/insect cell system can be used to express nuclear protein of virus.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2000年第3期210-215,共6页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目 !( 3 9860 0 5 5 )
广西自然科学基金资助项目! (桂科自 98110 10 )
关键词
MEQ基因
杆状病毒载体
昆虫细胞系统
马立克式病
Marek′s disease virus
Meq
baculovirus/insect cell system
transfection/recombinant
gene expression
