CAJ | 학술논문

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。
목적：제비유H5N1아형인금류감병독혈응소（HA）화기질단백1（M1）량충결구단백조장이성적H5아형류감병독양과립（ viruslike particles，VLPs）병검측기생물활성。방법구건동시포함 A／Indonesia／05／2005（ H5N1） HA 화 A／Anhui／01／2005（ H5N1） M1기인적중조전이재체pFBD-M1-HA，사선획득중조간립（중조간상병독질립） rBacmid-M1-HA후，전염Spodoptra frugiperda （Sf9）곤충세포，포장중조간상병독rBV-M1-HA；Western blot화간접면역형광감정HA화M1적표체；대량제비병순화후，재투사전경하관찰VLPs적형태결구；혈응시험검측VLPs적생물활성。결과응용곤충세포-간상병독표체계통표체H5N1 HA화M1량충결구단백가이고효조장산생류감VLPs，순화후적VLPs혈응효개위1024 HAU／50μl。결론성공이용A／Indonesia／05／2005（H5N1）래원HA화A／Anhui／01／2005（H5N1）래원M1량충결구단백，재간상병독표체계통중제비유교호생물활성적인금류감병독H5아형VLPs，위하일보연구인H5아형금류감VLPs역묘전정기출。
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.