Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36273
標題: 利用桿狀病毒表現系統製備具免疫原性的H1亞型流感病毒HA似病毒顆粒
Production of Immunogenic Baculovirus-Derived H1-subtype Influenza Hemagglutinin Virus-Like Particles
作者: 曾垣賓
Tseng, Yuan-Pin
關鍵字: influenza
流感病毒
Virus-like particles
VLP
Baculvirus
vaccine
Hemagglutinin
HA
H1N1
似病毒顆粒
桿狀病毒
疫苗
血球凝集素
出版社: 生物科技學研究所
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摘要: 流感似病毒顆粒為近年來新一代的流感疫苗,具有類似真實病毒顆粒的外形及抗原性、不具病毒核酸等特點。本研究使用桿狀病毒表現系統在Hi-5昆蟲細胞株表現H1N1亞型A/PR/8/34流感病毒株的血球凝集素蛋白(HA),經由血球吸附試驗證實感染的細胞可表現具有功能的HA膜蛋白,並且胞外的培養基具有血球凝集現象,顯示部分的HA分子被釋放到胞外區域。以病毒感染複數(MOI)最適化試驗,得到細胞以MOI = 0.01感染72小時具有最佳的胞外HA產量,約為64 HAU /50μl (1.2 μg/ml)。在20~60% 蔗糖梯度超高速離心及以動態光散射分析結果證實被釋放到胞外HA分子為顆粒型態,其浮力密度介於1.17~1.20 g/cm3,平均粒徑為174 ± 7 nm。進一步以穿透式電子顯微鏡及免疫金標定證實所觀察到的似病毒顆粒確實含有HA。另一方面,以介質輔助雷射脫附游離-飛行時間式質譜儀 (MALDI/TOF) 分析HA似病毒顆粒分層的組成,發現除了HA蛋白外,還有來自細胞的HSP70、beta-tubulin和cytoplasmic actin以及來自桿狀病毒的gp64蛋白,這些蛋白有可能參與HA似病毒顆粒的釋出過程。動物免疫實驗證實BALB/c小鼠在免疫兩次含0.2 μg或2 μg HA的似病毒顆粒後,皆可產生高力價的血球凝集抑制(HI)抗體 ( >150倍)。綜合上述結果顯示利用桿狀病毒表現系統在Hi-5昆蟲細胞株表現H1N1亞型A/PR/8/34流感病毒株的血球凝集素蛋白可以形成具有免疫原性的HA似病毒顆粒且具有開發成為流感似病毒顆粒疫苗的潛力。
Influenza virus-like particles (VLPs) are prominent influenza vaccine candidates because of their similar morphologies and immunogenicities to authentic virions and lack of viral genetic materials. In this study, hemagglutinin (HA) of influenza A/PR/8/34 (H1N1) virus was expressed as a membrane protein, which conferred the infected Hi-5 cells with the function to agglutinate erythrocytes. Strikingly, the hemagglutination was also detectable in the medium, which suggested some HA molecules were released into extracellular environment. Multiplicity of infection (MOI) optimization showed that cells were infected with 0.01 MOI at 72 hours post infection has the highest titer of extracellular HA for 64 HAU/ 50μl (1.2 μg/ml). 20-60% sucrose density gradient ultracentrifugation and dynamic laser scattering analysis were conducted to demonstrate that these extracellular HA molecules were released in a particle form with the buoyant densities between 1.17-1.20 g/cm3 and average diameter of 174 ± 7 nm. The existence of VLPs formed by HA (HA-VLPs) was further confirmed by electron microscopy and immunogold labeling. On the other hand, cell-derived HSP 70, cytoplasmic actin and beta-tubulin and baculovirus gp64 protein were MALDI /TOF-identified in the same fraction of HA-VLPs, which suggested that these proteins might involve in the budding process of HA-VLPs. Animal immunization demonstrated that two doses of VLPs containing 0.2 μg or 2 μg HA can elicit high titers of hemagglutination inhibition (HI) antibody (>150) in all vaccinated BALB/c mice. Taken together, these results demonstrates that using recombinant baculovirus expression system to express influenza virus A/PR/8/34 (H1N1) HA in Hi-5 insect cells is capable to produce immunogenic HA-VLPs and indicate that HA-VLPs represent another promise influenza VLP vaccine candidate.
URI: http://hdl.handle.net/11455/36273
其他識別: U0005-0702201201363200
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0702201201363200
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