摘要
将非典型犬瘟热病毒(Canine distemper virus,CDV)的核衣壳蛋白(nucleocapsid protein,N)基因克隆到杆状病毒表达系统供体质粒pFastBacHTA中,构建含N基因的重组供体质粒pFastBac-N,转化E.coli DH10Bac感受态细胞,经筛选获得含N基因的重组Bacmid DNA(rBacmid-N),脂质体法转染昆虫细胞Sf9,获得重组杆状病毒vBacmid-N。用vBacmid-N感染昆虫细胞Sf9,免疫印迹(Western blot)分析,在62kDa处出现一条特异蛋白条带,与重组N蛋白的理论值相符合;间接免疫荧光试验(indirect immunofluorescent assay,IFA)检测,vBacmid-N感染的昆虫细胞sf9出现特异绿色荧光。以纯化的重组N蛋白为抗原建立CDV抗体间接ELISA检测方法,犬CDV阳性血清A450大于0.40,而犬CDV阴性血清A450小于0.05,显示了良好的抗原特异性与稳定性。
The nucleocapsid（N） protein gene of an atypical Canine distemper virus（CDV） was cloned into pFastBacHTA donor plasmid.The recombinant donor plasmid pFastBac-N containing N gene was constructed and transformed into competent E.coli DH10Bac cells that were grown on LB plate containing 3 antibiotics.Recombinant Bacmid DNA（rBacmid-N） was obtained and used to transfect insect cells Sf9 with Lipofectamine to produce baculovirus vBacmid-N.The expressed recombinant N protein band of approximate 62 kDa was detected in Western blot.The recombinant vBacmid-N antigen was visualized in infected Sf9 cells in indirect immunofluorescence assay.Purified recombinant N protein was used to establish indirect ELISA for the detection of antibody against CDV.The absorbance values at 450 nm of all CD-positive serum samples were above 0.4 whereas CD-negative serum samples were below 0.05.
出处
《中国动物传染病学报》
CAS
2011年第5期21-26,共6页
Chinese Journal of Animal Infectious Diseases
基金
江苏省农业科技自主创新资金项目(SCX(11)2141)
关键词
犬瘟热病毒
核衣壳蛋白基因
昆虫细胞
真核表达
杆状病毒
Canine distemper virus（CDV）
nucleocapsid protein（N） gene
insect cells
eukaryotic expression
baculovirus
