以桿狀病毒呈現禽流感病毒之血球凝集素之研究－gp64蛋白之細胞質內區域對桿狀病毒之影響

碩士 國立清華大學 生物科技研究所 GH000934286 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-H...

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Bibliographic Details
Main Authors: 楊定罡, Yang, Ding-Gang
Other Authors: 黎耀基, 胡育誠, Lai, Yiu-Kay, Hu, Yu-Chen
Language:English
Chinese
Published: 2006
Subjects:
桿狀病毒
血球凝集素
禽流感
疫苗
avian influenza virus
baculovirus
hemagglutinin
surface display
vaccine
36
Avian flu
Online Access:http://nthur.lib.nthu.edu.tw/dspace/handle/987654321/29598
id ftnthuniv:oai:nthur.lib.nthu.edu.tw:987654321/29598
record_format openpolar
institution Open Polar
collection National Tsing Hua University Institutional Repository (NTHUR)
op_collection_id ftnthuniv
language English
Chinese
topic 桿狀病毒
血球凝集素
禽流感
疫苗
avian influenza virus
baculovirus
hemagglutinin
surface display
vaccine
36
spellingShingle 桿狀病毒
血球凝集素
禽流感
疫苗
avian influenza virus
baculovirus
hemagglutinin
surface display
vaccine
36
楊定罡
Yang, Ding-Gang
以桿狀病毒呈現禽流感病毒之血球凝集素之研究－gp64蛋白之細胞質內區域對桿狀病毒之影響
topic_facet 桿狀病毒
血球凝集素
禽流感
疫苗
avian influenza virus
baculovirus
hemagglutinin
surface display
vaccine
36
description 碩士 國立清華大學 生物科技研究所 GH000934286 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.
author2 黎耀基
胡育誠
Lai, Yiu-Kay
Hu, Yu-Chen
author 楊定罡
Yang, Ding-Gang
author_facet 楊定罡
Yang, Ding-Gang
author_sort 楊定罡
title 以桿狀病毒呈現禽流感病毒之血球凝集素之研究－gp64蛋白之細胞質內區域對桿狀病毒之影響
title_short 以桿狀病毒呈現禽流感病毒之血球凝集素之研究－gp64蛋白之細胞質內區域對桿狀病毒之影響
title_full 以桿狀病毒呈現禽流感病毒之血球凝集素之研究－gp64蛋白之細胞質內區域對桿狀病毒之影響
title_fullStr 以桿狀病毒呈現禽流感病毒之血球凝集素之研究－gp64蛋白之細胞質內區域對桿狀病毒之影響
title_full_unstemmed 以桿狀病毒呈現禽流感病毒之血球凝集素之研究－gp64蛋白之細胞質內區域對桿狀病毒之影響
title_sort 以桿狀病毒呈現禽流感病毒之血球凝集素之研究－gp64蛋白之細胞質內區域對桿狀病毒之影響
publishDate 2006
url http://nthur.lib.nthu.edu.tw/dspace/handle/987654321/29598
genre Avian flu
genre_facet Avian flu
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Coligan JE, Bierer BE, Margulies DH, Shevach EM, Strober W. 2005. Short protocols in immunology. New York: John Wiley & Sons. 11-42-11-43 p. Crawford J, Wilkinson B, Vosnesensky A, Smith G, Garcia M, Stone H, Perdue ML. 1999. Baculovirus-derived hemagglutinin vaccines protect against lethal influenza infections by avian H5 and H7 subtypes. Vaccine 17(18):2265-74. Dhar AK, Roux MM, Klimpel KR. 2001. Detection and quantification of infectious hypodermal and hematopoietic necrosis virus and white spot virus in shrimp using real-time quantitative PCR and SYBR Green chemistry. J Clin Microbiol 39(8):2835-45. Dolganiuc V, McGinnes L, Luna EJ, Morrison TG. 2003. Role of the cytoplasmic domain of the Newcastle disease virus fusion protein in association with lipid rafts. J Virol 77(24):12968-79. Edidin M. 2003. The state of lipid rafts: from model membranes to cells. Annu Rev Biophys Biomol Struct 32:257-83. Ernst W, Grabherr R, Wegner D, Borth N, Grassauer A, Katinger H. 1998. 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spelling ftnthuniv:oai:nthur.lib.nthu.edu.tw:987654321/29598 2023-05-15T15:34:38+02:00 以桿狀病毒呈現禽流感病毒之血球凝集素之研究－gp64蛋白之細胞質內區域對桿狀病毒之影響 Surface Display of Avian Influenza Virus Hemagglutinin on Baculovirus Envelope: Effects of Cytoplasmic Domain on Virus Properties 楊定罡 Yang, Ding-Gang 黎耀基 胡育誠 Lai, Yiu-Kay Hu, Yu-Chen 2006 155 bytes text/html http://nthur.lib.nthu.edu.tw/dspace/handle/987654321/29598 en zh_TW eng chi Alexander DJ. 2000. A review of avian influenza in different bird species. Vet Microbiol. 74(1-2):3-13. Barsoum J, Brown R, McKee M, Boyce FM. 1997. Efficient transduction of mammalian cells by a recombinant baculovirus having the vesicular stomatitis virus G glycoprotein. Hum Gene Ther 8(17):2011-8. Beigel JH, Farrar J, Han AM, Hayden FG, Hyer R, de Jong MD, Lochindarat S, Nguyen TK, Nguyen TH, Tran TH and others. 2005. Avian influenza A (H5N1) infection in humans. N Engl J Med 353(13):1374-85. Boublik Y, Di Bonito P, Jones IM. 1995. Eukaryotic virus display: engineering the major surface glycoprotein of the Autographa californica nuclear polyhedrosis virus (AcNPV) for the presentation of foreign proteins on the virus surface. Biotechnology (N Y) 13(10):1079-84. Brown EG. 2000. Influenza virus genetics. Biomed Pharmacother 54(4):196-209. Chazal N, Gerlier D. 2003. Virus entry, assembly, budding, and membrane rafts. Microbiol Mol Biol Rev 67(2):226-37, table of contents. Coligan JE, Bierer BE, Margulies DH, Shevach EM, Strober W. 2005. Short protocols in immunology. New York: John Wiley & Sons. 11-42-11-43 p. Crawford J, Wilkinson B, Vosnesensky A, Smith G, Garcia M, Stone H, Perdue ML. 1999. Baculovirus-derived hemagglutinin vaccines protect against lethal influenza infections by avian H5 and H7 subtypes. Vaccine 17(18):2265-74. Dhar AK, Roux MM, Klimpel KR. 2001. Detection and quantification of infectious hypodermal and hematopoietic necrosis virus and white spot virus in shrimp using real-time quantitative PCR and SYBR Green chemistry. J Clin Microbiol 39(8):2835-45. Dolganiuc V, McGinnes L, Luna EJ, Morrison TG. 2003. Role of the cytoplasmic domain of the Newcastle disease virus fusion protein in association with lipid rafts. J Virol 77(24):12968-79. Edidin M. 2003. The state of lipid rafts: from model membranes to cells. Annu Rev Biophys Biomol Struct 32:257-83. Ernst W, Grabherr R, Wegner D, Borth N, Grassauer A, Katinger H. 1998. Baculovirus surface display: construction and screening of a eukaryotic epitope library. Nucleic Acids Res 26(7):1718-23. Facciabene A, Aurisicchio L, La Monica N. 2004. Baculovirus vectors elicit antigen-specific immune responses in mice. J. Virol. 78(16):8663-8672. Ghosh S, Parvez MK, Banerjee K, Sarin SK, Hasnain SE. 2002. Baculovirus as mammalian cell expression vector for gene therapy: an emerging strategy. Mol Ther 6(1):5-11. Grabherr R, Ernst W, Oker-Blom C, Jones I. 2001. Developments in the use of baculoviruses for the surface display of complex eukaryotic proteins. Trends Biotechnol 19(6):231-6. Hammonds J, Chen X, Ding L, Fouts T, De Vico A, zur Megede J, Barnett S, Spearman P. 2003. Gp120 stability on HIV-1 virions and Gag-Env pseudovirions is enhanced by an uncleaved Gag core. Virology 314(2):636-49. Hofmann C, Sandig V, Jennings G, Rudolph M, Schlag P, Strauss M. 1995. Efficient gene-transfer into human hepatocytes by baculovirus vectors. Proc. Natl. Acad. Sci. U.S.A. 92(22):10099-10103. Hu Y-C, Tsai C-T, Chung Y-C, Lu J-T, Hsu JT-A. 2003. Generation of chimeric baculovirus with histidine-tags displayed on the envelope and its purification using immobilized metal affinity chromatography. ENZYME MICROB TECH 33:445-452. Hu YC, Luo YL, Ji WT, Chulu JL, Chang PC, Shieh H, Wang CY, Liu HJ. 2006. Dual expression of the HA protein of H5N2 avian influenza virus in a baculovirus system. J Virol Methods 135(1):43-8. Hughes GJ, Smith JS, Hanlon CA, Rupprecht CE. 2004. Evaluation of a TaqMan PCR assay to detect rabies virus RNA: influence of sequence variation and application to quantification of viral loads. J Clin Microbiol 42(1):299-306. Jarvis DL, Garcia A, Jr. 1994. Biosynthesis and processing of the Autographa californica nuclear polyhedrosis virus gp64 protein. Virology 205(1):300-13. Jin H, Leser GP, Zhang J, Lamb RA. 1997. Influenza virus hemagglutinin and neuraminidase cytoplasmic tails control particle shape. EMBO J 16(6):1236-47. Kaba SA, Hemmes JC, van Lent JW, Vlak JM, Nene V, Musoke AJ, van Oers MM. 2003. Baculovirus surface display of Theileria parva p67 antigen preserves the conformation of sporozoite-neutralizing epitopes. Protein Eng 16(1):73-8. Kaikkonen MU, Raty JK, Airenne KJ, Wirth T, Heikura T, Yla-Herttuala S. 2006. Truncated vesicular stomatitis virus G protein improves baculovirus transduction efficiency in vitro and in vivo. 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J Virol 77(11):6265-73. 桿狀病毒 血球凝集素 禽流感 疫苗 avian influenza virus baculovirus hemagglutinin surface display vaccine 36 2006 ftnthuniv 2014-12-17T20:01:49Z 碩士 國立清華大學 生物科技研究所 GH000934286 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. Other/Unknown Material Avian flu National Tsing Hua University Institutional Repository (NTHUR)

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