鱼类神经坏死病病毒衣壳蛋白在昆虫细胞中的表达及其特性
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国家高技术研究发展计划


A study on the biological character of capsid protein expressed in insect cells from fish viral nervous necrosis virus
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    摘要:

    利用RT-PCR技术获得病毒性神经坏死病毒0603株的衣壳蛋白基因,将其插入到杆状病毒Bac-To-Bac表达系统的pFastBacⅠ质粒中,构建了pFastBac-cp质粒。转化DH10Bac大肠杆菌后获得重组穿梭载体Bacmid-cp,脂质体介导将其转染Sf9细胞产生有感染性的重组杆状病毒AcNPV-cp。利用AcNPV-cp感染Sf9细胞后,SDS-PAGE分析可见大小约为37ku的特异性蛋白带,Western-blotting分析发现,其可以与病毒性神经坏死病毒阳性血清反应出现特异性的杂交带。试验结果表明,AcNPV-cp在Sf9细胞中成功地表达了病毒性神经坏死病毒的衣壳蛋白,其具有良好的免疫学活性。负染电镜观察发现,CP蛋白可自行装配成病毒样颗粒,其大小形态类似于病毒性神经坏死病毒。制备超薄切片后电镜观察发现,CP蛋白自行装配成的病毒样颗粒呈晶格状排列在细胞质中。为研制有效防控鱼类病毒性神经坏死病的新型颗粒性疫苗奠定了基础。

    Abstract:

    Viral nervrou necrosis virus (VNNV) causes the pathogenic effects in the nerve tissues of many marine fish, which caused great economic loss in mariculture of many countries. In this study, the capsid protein (CP) gene of VNNV 0603 Strain was amplified successfully by reverse-transcription polymerase chain reaction (RT-PCR), and inserted into plasmid pFastBacⅠof the Baculovirus expression system Bac-To-Bac to develop plasmid pFastBac-cp. Through homologous recombination between pFastBac-cp and Bacmid which was the modified genome of Autogragha california nuclear polyhedrosis virus (AcNPV), the recombinant shuttle vector named Bacmid-cp was obtained in E. coli DH10Bac. After tranfection with Cellfectin Reagent, recombinant Baculovirus (AcNPV-cp) was formed in Sf9 cells. The specific 37kD protein band and the hybridizing reaction between expressed protein and positive serum for VNNV were showed by analysis of SDS-PAGE and Western blotting, which indicated that VNNV CP protein had been expressed in insect cells Sf9 with immunological activity. The self-assembled virus-like particles (VLPs) were observed in the negative staining CP crude extract and in the ultra thin section of infected Sf9 cells with electron microscopy, which were similar to VNNV whole virion, and became a crystal array in cytoplasm. On the base of present study, the particle vaccine of VNNV would be developed and put into practice in the near future.

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罗卫,田飞焱,刘荭.鱼类神经坏死病病毒衣壳蛋白在昆虫细胞中的表达及其特性[J].水产学报,2008,32(4):651~658

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  • 收稿日期:2007-05-29
  • 最后修改日期:2007-11-27
  • 录用日期:2008-05-28
  • 在线发布日期: 2008-07-07
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