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目的构建同时含有禽流感H5N1疫苗株NIBRG-14的血凝素(Hemagglutinin,HA)、神经氨酸酶(Neurami-nidase,NA)及基质蛋白(Matrix protein,M1)3种基因的重组杆状病毒,并研究其表达产物的生物学特性。方法利用RT-PCR法从禽流感H5N1疫苗株NIBRG-14中扩增HA、NA及M1基因片段,并同时亚克隆至杆状病毒穿梭质粒pFastBacDual中,构建重组杆状病毒穿梭质粒,转染Sf9昆虫细胞,包装重组杆状病毒Bacmid-HA-NA-M1a。Westernblot法和血凝试验检测HA、NA及M1蛋白在Sf9细胞中的表达;表达的重组蛋白经超速离心浓缩及蔗糖密度梯度离心纯化后,透射电镜观察病毒样颗粒(Virus-like particles,VLPs)的形态结构,并通过单向免疫扩散(Single radialimmunodiffusion,SRID)试验检测HA的含量。结果已成功构建了重组杆状病毒,该病毒可同时表达HA、NA及M1蛋白,3种蛋白可自行组装成VLPs,并分泌至细胞培养上清中,血凝效价可达1∶64;重组杆状病毒表达的蛋白形成的VLPs与NIBRG-14标准抗血清形成沉淀环,浓缩及纯化后的重组蛋白HA含量分别为36和27μg/ml;透射电镜观察可见直径约100 nm的典型流感VLPs。结论构建的重组杆状病毒可表达VLPs,为禽流感新型疫苗的研制奠定了基础。
Objective To construct a recombinant baculovirus that contains three genes of hemagglutinin (HA), neuraminidase (NA) and matrix protein (M1) of NIBRG-14 strain of bird flu H5N1 vaccine strain, , And study the biological characteristics of its expression product. Methods The HA, NA and M1 gene fragments were amplified by RT-PCR from the H5N1 vaccine strain NIBRG-14 and subcloned into the shuttle plasmid pFastBacDual to construct a recombinant shuttle baculovirus vector and transfected into Sf9 Insect cells, packaging recombinant baculovirus Bacmid-HA-NA-M1a. Western blotting and hemagglutination assay were used to detect the expression of HA, NA and M1 protein in Sf9 cells. The expressed recombinant protein was purified by ultracentrifugation and sucrose density gradient centrifugation. Transmission electron microscopy was used to observe the expression of the virus-like particles (VLPs) The morphological structure of HA was detected and the content of HA was detected by single radial immunodiffusion (SRID). Results Recombinant baculovirus was successfully constructed. The virus could express HA, NA and M1 protein simultaneously. The three proteins could be assembled into VLPs by themselves and secreted into the cell culture supernatant with the titer of 1:64. The recombinant protein expressed by recombinant baculovirus formed a precipitation loop with NIBRG-14 standard antiserum, and the HA content of recombinant protein was 36 and 27μg / ml respectively. Transmission electron microscopy showed typical influenza VLPs with a diameter of about 100 nm . Conclusion The constructed recombinant baculovirus can express VLPs and lay a foundation for the development of a novel vaccine against avian influenza.