高致病性人禽流感 H5N1病毒 HA 与 M1蛋白共表达制备新型病毒样颗粒

Characterizaiton of virus-like particles prepared by Bac-to-Bac system co-expressing HA and M1 ;protein of highly pathogenic avian influenza H5N1

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.