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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37ku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。
The capsid protein gene of Vero virus 0603 was obtained by RT-PCR and inserted into the pFastBacI plasmid of Bac-To-Bac expression system of baculovirus to construct pFastBac-cp plasmid. The recombinant shuttle vector Bacmid-cp was obtained after transformation of DH10Bac E.coli. Lipofectamine was transfected into Sf9 cells to produce infectious recombinant baculovirus AcNPV-cp. After Sf9 cells were infected with AcNPV-cp, a specific protein band of about 37ku was found by SDS-PAGE analysis. Western blotting showed that it could specifically hybridize with the positive serum of virus-induced necrosis virus. The experimental results showed that AcNPV-cp successfully expressed the capsid protein of the viral necrotic virus in Sf9 cells, which has good immunological activity. Negative-staining electron microscopy showed that the CP protein could assemble into virus-like particles by itself, and its size and shape were similar to those of the virus-induced necrosis virus. After the preparation of ultra-thin sections by electron microscopy, we found that the virus-like particles self-assembled by CP protein are arranged in the cytoplasm in the form of lattices. Which laid the foundation for the development of a new type of particle vaccine that effectively prevents and controls the viral viral necrosis of the fish.