摘要
目的:制备由H5N1亚型人禽流感病毒血凝素(HA)和基质蛋白1(M1)两种结构蛋白组装而成的H5亚型流感病毒样颗粒( viruslike particles,VLPs)并检测其生物活性。方法构建同时包含 A/Indonesia/05/2005( H5N1) HA 和 A/Anhui/01/2005( H5N1) M1基因的重组转移载体pFBD-M1-HA,筛选获得重组杆粒(重组杆状病毒质粒) rBacmid-M1-HA后,转染Spodoptra frugiperda (Sf9)昆虫细胞,包装重组杆状病毒rBV-M1-HA;Western blot和间接免疫荧光鉴定HA和M1的表达;大量制备并纯化后,在透射电镜下观察VLPs的形态结构;血凝试验检测VLPs的生物活性。结果应用昆虫细胞-杆状病毒表达系统表达H5N1 HA和M1两种结构蛋白可以高效组装产生流感VLPs,纯化后的VLPs血凝效价为1024 HAU/50μl。结论成功利用A/Indonesia/05/2005(H5N1)来源HA和A/Anhui/01/2005(H5N1)来源M1两种结构蛋白,在杆状病毒表达系统中制备有较好生物活性的人禽流感病毒H5亚型VLPs,为下一步研究人H5亚型禽流感VLPs疫苗奠定基础。
Objective To express and characterize the virus-like particles( VLPs) of H5 subtype containing of hemagglutinin ( HA ) and matrix 1 ( M1 ) protein by using Baculovirus-insect cells .Methods Full length genes encoding HA protein from the A/Indonesia/05/2005(H5N1) strain and the M1 protein from the A/Anhui/01/2005 ( H5N1 ) strain were cloned into a baculovirus expression vector to construct pFBD-M1-HA.The expression of HA and M1 proteins were detected by Western blot and indirect immunoflu-orescence after the transfection of Spodoptra frugiperda (Sf9) insect cells with recombinant baculovirus.Pu-rified VLPs were analyzed by SDS-PAGE and visualized with transmission electron microscope.The biologi-cal activity of purified VLPs was detected by hemagglutination test.Results The HA and M1 proteins of H5 subtype expressed by baculovirus-insect cells could be self-assembled into the functional mature VLPs.The hemagglutination titer of VLPs was as high as 1024 HAU/50μl.Conclusion The H5 subtype VLPs as pre-pared in this study would pave a way for the development of a candidate recombinant A ( H5) vaccine.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2015年第4期286-291,共6页
Chinese Journal of Microbiology and Immunology
基金
传染病防治重大专项(2013 ZX10004001-004)
国家自然科学基金(81302607)