| 犬瘟热病毒核衣壳蛋白基因在昆虫细胞中的表达及其抗原性分析 |
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| 引用本文： | 毕振威,王永山,范红结.犬瘟热病毒核衣壳蛋白基因在昆虫细胞中的表达及其抗原性分析[J].中国兽医寄生虫病,2011,19(5):21-26. |
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| 作者姓名： | 毕振威 王永山 范红结 |
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| 作者单位： | 1. 江苏省农业科学院兽医研究所农业部动物疫病诊断与免疫重点开放实验室国家兽用生物制品工程技术研究中心,南京,210014
2. 南京农业大学动物医学院,南京,210095 |
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| 基金项目： | 江苏省农业科技自主创新资金项目(SCX |
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| 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。
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| 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 |
EXPRESSION IN INSECT CELLS AND ANTIGENIC ANALYSIS OF RECOMBINANT NUCLEOCAPSID PROTEIN OF CANINEDISTEMPER VIRUS |
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| BI Zhen-wei,WANG Yong-shan,FAN Hong-jie.EXPRESSION IN INSECT CELLS AND ANTIGENIC ANALYSIS OF RECOMBINANT NUCLEOCAPSID PROTEIN OF CANINEDISTEMPER VIRUS[J].Chinese Journal of Veterinary Parasitology,2011,19(5):21-26. |
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| Authors: | BI Zhen-wei WANG Yong-shan FAN Hong-jie |
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| Institution: | BI Zhen-wei1,2,WANG Yong-shan1,FAN Hong-jie2(1.National Center for Engineering Research of Veterinary Bio-products,Key Laboratory of Animal Diseases Diagonostic and Immunology,Ministry of Agriculture,Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China,2.College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China) |
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| Abstract: | 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. |
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| Keywords: | Canine distemper virus（CDV） nucleocapsid protein（N） gene insect cells eukaryotic expression baculovirus |
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