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目的:应用重组杆状病毒表达系统制备由HA、NA、M1和M2蛋白组成的H5N1高致病性禽流感病毒样颗粒,为研究H5N1高致病性禽流感疫苗奠定基础。方法:构建能共表达A/chicken/Jilin/2003(H5N1)禽流感病毒血凝素(HA)和神经氨酸酶(NA)、A/PR/8/34(H1N1)流感病毒基质蛋白(M1)和离子通道蛋白(M2)的2个二元重组杆状病毒,共同感染HighFive细胞,同时表达HA、NA、M1和M2蛋白,使这4种蛋白在感染的细胞内自主组装成病毒样颗粒。经差速离心和蔗糖密度梯度超速离心收获病毒样颗粒,通过Western印迹鉴定病毒样颗粒的组成,透射电镜观察病毒样颗粒形态,血凝试验测定病毒样颗粒的活性。结果:HA、NA、M1、M2蛋白在昆虫细胞中共表达,并组装成病毒样颗粒;电镜观察到病毒样颗粒的形态与流感病毒一致,直径约80 nm;血凝试验显示该病毒样颗粒具有凝集鸡红细胞的活性。结论:应用该方法可以制备流感病毒样颗粒,为H5N1流感疫苗研究提供了可行方案。
OBJECTIVE: To prepare H5N1 HPAI-like particles composed of HA, NA, M1 and M2 proteins by using recombinant baculovirus expression system, which laid the foundation for the study of H5N1 HPAI vaccine. Methods: The recombinant adenovirus vector was constructed to co-express HA / NA and A / PR / 8/34 (H1N1) influenza virus matrix protein ) And ion channel protein (M2) were co-infected with HighFive cells and HA, NA, M1 and M2 proteins were co-infected. These four proteins were self-assembled into virus-like particles in infected cells . Virus-like particles were harvested by differential centrifugation and sucrose density gradient ultracentrifugation. The composition of the virus-like particles was identified by Western blotting. The shape of the virus-like particles was observed by transmission electron microscopy and the activity of the virus-like particles was determined by the hemagglutination assay. Results: The HA, NA, M1 and M2 proteins were co-expressed in insect cells and assembled into virus-like particles. The morphology of the virus-like particles was consistent with the influenza virus by electron microscopy and the diameter was about 80 nm. The hemagglutination test showed that the virus- Agglutination of chicken erythrocyte activity. Conclusion: The method can be used to prepare influenza virus-like particles, providing a feasible solution for the study of H5N1 influenza vaccine.