应用HA、NA、M1和M2蛋白制备H5N1高致病性禽流感病毒样颗粒

Highly Pathogenic H5N1 Avian Influenza Virus-Like Particles Comprised of the HA,NA,M1 and M2 Proteins

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 develop recombinant vaccine for avian influenza of the H5N1 subtype,we expressed virus-like particles（VLP） consisting of four structural proteins of influenza virus in insect cells.Methods： Influenza VLP were produced in insect cells co-infected with two recombinant baculoviruses,one expressing the hemagglutinin（HA） and the neuraminidase（NA） of A/chicken/Jilin/2003（H5N1） virus,another expressing the matrix protein（M1） and the ion-channel protein（M2） of A/PR/8/34（H1N1） virus.VLP were purified by differential centrifugation and sucrose density gradient ultra-centrifugation.The purified VLP preparation was confirmed by Western blot analysis.In addition,negatively stained preparations of VLP were examined by transmission electron microscopy.The titers of VLP were calculated by haemagglutination test.Results： Upon infection of insect cells with recombinant baculoviruses,HA,NA,M1 and M2 proteins were co-expressed in the infected cells,self-assembled,and released into the culture medium as VLP of about 80 nm in diameter.VLP exhibited functional characteristics of influenza virus including hemagglutination activities.Conclusion： Thus,VLP represent a potential strategy for the development of human vaccines against avian influenza H5N1 viruses.