以桿狀病毒為基因載體於哺乳動物細胞內組裝D型肝炎類病毒顆粒之研究

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研究生：	王貴君 Kuei-Chun Wang
論文名稱：	以桿狀病毒為基因載體於哺乳動物細胞內組裝D型肝炎類病毒顆粒之研究 Baculovirus as a highly efficient gene delivery vector for the assembly of hepatitis delta virus-like particles in mammalian cells
指導教授：	張大慈 Margaret Dah-Tsyr Chang 胡育誠 Yu-Chen Hu
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 分子與細胞生物研究所 Institute of Molecular and Cellular Biology
論文出版年：	2004
畢業學年度：	92
語文別：	英文
論文頁數：	77
中文關鍵詞：	桿狀病毒、D型肝炎病毒、類病毒顆粒、哺乳動物細胞表現系統
外文關鍵詞：	baculovirus, hepatitis delta virus, virus-like particle, mammalian cell expression system
相關次數：	點閱：3 下載：0
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摘要
桿狀病毒 (Baculovirus)近年來逐漸被發現具有開發為哺乳動物細胞基因傳導載體之潛力。本研究中，我們進一步將桿狀病毒的應用延伸到D型肝炎病毒 (Hepatitis Delta virus)類病毒顆粒 (VLP)的組裝上。首先，建構兩株重組桿狀病毒Bac-GD和Bac-GB，分別帶有L-HDAg、HBsAg及綠色螢光蛋白 (eGFP)的基因並受哺乳動物細胞啟動子調控。藉著流式細胞儀分析最佳的轉導條件，我們發現以100 μl (MOI=100)的未濃縮病毒在25 □C 的D-PBS環境溶液下進行感染肝癌細胞株 (HuH-7) 6 h，可達到最好的轉導效果。透過同步定量PCR的結果，我們也證明了桿狀病毒的確比其他轉染方法 (如:微脂粒和磷酸鈣共同沉澱法)具有更高的基因傳導效率；即使在高細胞密度下，桿狀病毒的高轉導效率仍不受影響。為了提高表現量，我們使用EGTA及丁酸鈉來增強病毒的轉導效率以及基因表現；除此之外，我們也進行重複感染以求在非溶裂的情況延長基因表現的時間。由桿狀病毒轉導肝細胞所表現的L-HDAg不僅特性皆與真實病毒蛋白相同，同時成功的與HBsAg組裝成類病毒顆粒並分泌至培養液中。透過了氯化銫梯度的等密度超高速離心，我們成功的從培養液中純化出D型肝炎病毒類病毒顆粒，並於穿透式電子顯微鏡觀察下發現類病毒顆粒之形態及大小，皆與真實病毒相似。總括來說，桿狀病毒作為D型肝炎病毒基因傳導載體的研究，提供了一個更有效率研究病毒機制的方法。除此之外，本系統的建立更有利於哺乳動物細胞中大量生產類病毒顆粒，做為醫學研究之用。

ABSTRACT
Baculovirus has been employed as an efficient gene delivery vector into mammalian cells for a variety of purposes. In this study, we further expanded the applications of baculovirus as a tool to study the assembly of hepatitis delta virus-like particle. To this end, two recombinant baculoviruses were constructed to express large hepatitis delta antigen and hepatitis B surface antigen under the regulation of mammalian promoters. Simple and efficient gene transduction into hepatoma cell lines (80-90% as determined by flow cytometry) and high level transgene expression (in the order of microgram per million cells) were achieved by incubating the cells with unconcentrated virus supplemented with Dulbecco’s phosphate-buffered saline at 25 □C for 6 h. Quantitative real-time PCR (Q-PCR) analyses quantitatively revealed that baculovirus transduction was more efficient than plasmid transfection with respect to DNA uptake and DNA transport to the nucleus, even for cells grown to near confluency. HDV antigen expression was enhanced by EGTA pretreatment and addition of sodium butyrate in culture medium and prolonged by superinfection. L-HDAg was correctly isoprenylated and localized to nucleus of the transduced cells as the authentic protein. Upon co-transduction, HDV-like particle could be assembled and secreted into medium, which were further purified from the culture medium by isopycnic ultracentrifugation and visualized by electron microscopy. This study demonstrated that baculovirus transduction is a suitable method alternative to plasmid transfection for efficient formation of HDV-like particles. This system may be also a useful tool in investigating cellular processes involved in HDV assembly and in producing large amount of HDV-like particles in bioreactor for medical research.

Contents
Chapter 1 INTRODUCTION………………………...……………………………….1
1-1 Baculovirus biology…………………………………………………………….1
1-2 Recombinant baculovirus as mammalian cell gene-delivery vector……………4
1-3 Hepatitis Delta virus (HDV)……………………………………………………5
1-4 Motivations……………………………………………………………8
Chapter 2 MATERIALS & METHODS……………………………………………9
2-1 Generation of recombinant baculovirus………………………………………...9
2-1-1 BAC-TO-BAC® Bacculovirus Expression System………………………….9
2-1-2 Construction of Bac-GD and Bac-GB vectors……………………………10
2-1-3 Transposition……………………………………………………………...11
2-1-4 Isolation of bacmid……………………………………………………….11
2-1-5 Transfection ……………….……………………………………………..12
2-1-6 Virus amplification and titration………………………………………….12
2-2 Cell culture and medium………………………………………………………13
2-3 Baculovirus transduction and transfection….…………………………………14
2-3-1 Baculovirus transduction…………………………………………………14
2-3-2 Transfection by liposome and CaPi-DNA co-precipitation…….………...14
2-4 SDS-PAGE and Western blot……………………...…………………………..15
2-5 Flow cytometry………………………………………………………………..16
2-6 Real-time PCR………………………………………………………………...16
2-7 Immunofluorescence…………………………………………………………..17
2-8 Isolation of antigens by ultracentrifugation…………………………………...18
2-8-1 Medium concentration by sucrose cushion……………………………….18
2-8-2 Partial purification of HDV-like particles by CsCl gradient……………...19
IV
2-9 Transmission electron microscopy…………………………………………….19
Chapter 3 RESULTS……………………………………..………….………………23
3-1 Baculovirus mediated gene transduction into mammalian cells………………23
3-2 Optimization of transduction conditions for baculovirus transduction………..24
3-2-1 Dependence of transduction efficiency on cell type and efficiency……...25
3-2-2 Effect of surrounding solution on transduction efficiency……………….25
3-2-3 Depenedence of transduction efficiency on incubation time………….….26
3-2-4 Effect of different temperatures on transduction efficiency……………...27
3-3 Comparison of gene transfer methods………………………………………....28
3-3-1 Comparison of efficiency by baculovirus transduction, lipofection
and CaPi-DNA co-precipitation………..………………………………...28
3-3-2 Effect of confluence………………………………………………………29
3-4 Enhancement and prolongation of baculovirus mediated gene transduction….29
3-4-1 Effect of sodium butyrate on protein expression level…………………...29
3-4-2 Transient disruption of cell-cell junction by EGTA……………………30
3-4-3 Superinfection for prolonged protein expression………………………31
3-5 Characterization of L-HDAg and HBsAg by baculovirus transduction………33
3-5-1 Localization of L-HDAg in nuclei………………………………………..33
3-5-2 Isoprenylation analysis of L-HDAg………………………………………33
3-5-3 Formation and secretion of HDV-like particles by co-infection………….34
3-6 Characterization and electron microscopic examination of HDV-like
particles purified from Baculovirus-transduced HuH-7 cells…….……..……..35
3-6-1 Purification of HDV-like particles by ultracentrifugation………………..35
3-6-2 TEM analysis of particles isolated from culture medium………………...36
3-6-3 Secretion efficiency of HDV-like particles……………………………….38
Chapter 4 DISCUSSION…..…..……………………………………………………58
V
Chapter 5 CONCLUSIONS……..…………………...……………………………...67
Chapter 6 FUTURE WORK…………...……………..……………………………..68
REFERENCES…………………..…………………………………………………...71

                                

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