流行感冒病毒屬於正黏液病毒科的套膜病毒。病毒的結構主要由HA，NA，M1，和M2這四個蛋白所組成。其中，HA為穿透膜的醣蛋白，並以三聚體的形式存在於外套膜上。由於病毒表面的棘蛋白主要都是HA，因此它是病毒引起抗體反應的主要抗原；另外，HA可以辨別宿主細胞膜上含sialic acid 的受體並與之結合，所以它也決定了所要感染宿主的種類。在本篇論文中，利用重組桿狀病毒在Sf9昆蟲細胞中表現A/WSN/33(H1N1) 和 A/Tailand/1(KAN-1)/2004(H5N1) 的HA，而後藉由PNGaseF 和Trypsin作用來確認Sf9所產生的HA具有醣化修飾且保有原來的結構，最後利用紅血球凝集實驗來確認其生物活性。經由實驗，可以發現不論是表現完整的HA或是HA的細胞外區域，他們仍主要停留在細胞內，只有少量HA的細胞外區域蛋白才會分泌出胞外。並且，進一步利用固定化金屬色層分析法純化重組的HA細胞外區域。先前的研究發現，單獨表現流感病毒的M1蛋白即可以產生類病毒顆粒。然而， M2蛋白的細胞膜區域也與病毒顆粒的組裝與形態有關。所以，我們不僅表現只含有M1的類病毒顆粒；並在M2蛋白的N端接上綠色螢光蛋白，藉由和M1同時表現，以構築含綠色螢光的流感類病毒顆粒。而這兩種似病毒顆粒將以蔗糖梯度分離法進行初步的分離，最後藉由穿透式電子顯微鏡來觀察其型態。

Influenza types A virus are envelope viruses, belonging to the Orthomyxoviridae family. The viral envelopes consist mainly four structure proteins (HA, NA, M1, and M2) and the plasma membrane-derived lipid biolayers. HA is the major spike protein on the viral surface and is involved in host cell binding, fusion and host cell tropism, and is the primary target antigen to elicit protective immune responses. In this study, recombinant HA proteins of the influenza A/WSN/33(H1N1) and A/Tailand/1(KAN-1)/2004(H5N1) strains were cloned and produced using baculovirus-insect cell expression system. Baculovirus-expressed H1HA and H5HA proteins were N-linked glycosylated, typrsin-sensitive for protease cleavage, and functionally agglutinated the red blood cells. Full-length and ectodomain H1HA and H5HA were effectively expressed in insect cells but not led to predominant secretion to culture medium. Finally, using immobilized metal ion affinity chromatography (IMAC) column to purify the recombinant HA ectodomain. Recombinant virus-like particles were obtained by baculovirus expression of M1 alone, as explained by sucrose gradient and transmission electron microscopy. Fluorescent virus-like particles were further constructed by co-expression of M1 and the M2 fused with EGFP at N-terminus.

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