血球凝集素（Hemagglutinin, HA）是構成禽流感病毒（AIV）外套膜的重要蛋白之一。因此，此研究最主要的目標便是利用兩種不同的細胞質內區域(cytoplasmic domain, CTD)來修飾HA，將HA呈現在桿狀病毒的外套膜上，並同時探討這兩種細胞質內區域對HA的呈現效果與桿狀病毒特性的影響。另外，本研究進一步地評估了以這種假性(pseudotyped)桿狀病毒作為一種創新疫苗來預防禽流感的可能性。為了達到這樣的目的，本研究中建構了兩株重組桿狀病毒。其中Bac-HA可表現經由HA CTD 修飾的HA，而另一株Bac-HA64可表現經由gp64 CTD修飾的HA（gp64 為桿狀病毒自身的封套蛋白）。之後分別以這兩種桿狀病毒來感染Sf9細胞，並以西方點墨法及共軛焦顯微鏡來偵測其表現及分佈的情形。證實這兩種經過修飾的HA均可在Sf9細胞內表現並分佈在其細胞膜上。同時藉由免疫金的標定，Bac-HA及Bac-HA64皆可分別將其表現之HA呈現在其外套膜上。然而，藉由凝膠電泳(SDS-PAGE)及西方點墨法的分析證實，在Bac-HA64的外套膜上帶有比Bac-HA還要多的HA含量，這樣的結果暗示著gp64 CTD可更有效的將HA嵌入桿狀病毒之外套膜中。而利用流式細胞儀及同步定量PCR的偵測發現，擁有較高HA含量的Bac-HA64可提升桿狀病毒進入多種哺乳動物細胞的能力，並促進轉殖基因的表現。另外，在經過37℃的置放後，Bac-HA64擁有與控制組的桿狀病毒相似的穩定性。另外，以Bac-HA64來免疫BALB/c小鼠後發現，其所誘發之免疫反應比起對照組的Bac-HA，有較高的血球凝集抑制(HI)的效果。這些實驗結果共同地證實了gp64 CTD可以更有效率的呈現HA，進而促進病毒的侵入與基因表現的能力，並且提高了免疫的效果。因此，Bac-HA64將可作為一個有潛力的新型疫苗來預防禽流感。

Hemagglutinin (HA) is the major immunogen on the envelope of avian influenza virus (AIV). To examine how the choice of cytoplasmic tail domain (CTD) affected the display of HA on baculoviral envelope and baculovirus properties, and evaluate the feasibility of HA-pseudotyped baculovirus as a vaccine against AIV infection, we constructed two pseudotyped baculoviruses: Bac-HA expressing chimeric HA with the CTD derived from HA, and Bac-HA64 expressing chimeric HA with the CTD derived from baculovirus envelope protein gp64. After infection with Bac-HA or Bac-HA64, HA with either CTD was anchored on the plasma membrane of Sf9 cells as revealed by confocal microscopy. Immunogold electron microscopy demonstrated that both Bac-HA and Bac-HA64 displayed HA on the viral surface. However, SDS-PAGE and Western blot analyses of purified viruses unraveled that a significantly higher amount of HA was incorporated into Bac-HA64 than into Bac-HA. In comparison with Bac-HA, Bac-HA64 significantly improved the gene delivery and transgene expression in mammalian cells as determined by quantitative real-time PCR and flow cytometry. Immunization of BALB/c mice with Bac-HA64 elicited significantly higher hemagglutination inhibition titers than Bac-HA and the negative controls. These data collectively confirmed that gp64 CTD, in comparison with HA CTD, endowed more efficient HA incorporation into baculovirus, more efficient transgene delivery and expression, as well as elevated immunogenicity. In conclusion, this is the first report demonstrating that the choice of CTD has a tremendous impact on baculovirus property and vaccine efficacy. The baculovirus-based vaccine may hold great promise as a novel platform to prevent avian flu epidemic and be envisaged as an alternative option in the priming-boosting vaccination scheme.

Chapter 1 INTRODUCTION 1
1.1 Avian influenza virus 1
1.2 Hemagglutinin of influenza virus 2
1.3 Baculovirus expression system 3
1.4 Baculovirus surface display 4
1.5 Motivation 5
Chapter 2 MATERIALS AND METHODS 8
2.1 Generation of recombinant baculovirus 8
2.1.1 BAC-TO-BAC® Baculovirus Expression System . 8
2.1.2 Construction of pBac-HA and pBac-HA64 vectors 8
2.1.3 Transposition 10
2.1.4 Transfection 11
2.1.5 Virus production and titration 11
2.2 Cells and culture medium 12
2.3 Virus concentration and purification 12
2.4 Western blot analysis 13
2.5 Confocal microscopy 13
2.6 Immunogold electron microscopy 14
2.7 Mammalian cell transduction and measurement of transducing titer 15
2.8 Flow cytometry 15
2.9 Real-time PCR 16
2.10 Immunization 16
2.11 Hemagglutination inhibition titration 17
Chapter 3 RESULT 21
3.1 Recombinant baculovirus construction 21
3.2 Expression of recombinant HA 22
3.3 Purification of recombinant baculovirus 23
3.4 Incorporation of recombinant HA on baculovirus 23
3.5 Characterization of recombinant baculovirus 23
3.6 Enhanced baculoviral transduction by the gp64 CTD 24
3.7 Improved virus entry ability in Bac-HA64 25
3.8 The amounts of HA incorporated influenced the baculovirus stability 26
3.9 Potentials of HA-pseudotyped baculoviruses as vaccine 26
Chapter 4 DISCUSSION 38
Chapter 5 FUTURE WORK 45
REFERENCE 47

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