Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90043
標題: Expressing Infectious bursal disease virus vaccine by coupling Bamboo mosaic virus vector and plant transgenesis
應用竹嵌紋病毒載體及基因轉殖在植物體表達雞華氏囊病毒疫苗蛋白
作者: 袁琴雅
Chin-Ya Yuan
關鍵字: 無;no
引用: 詹明才。1992。農桿菌轉殖水稻系統的建立。國立台灣大學農藝學研究所博士論文。 Alvarez, M.L., Pinyerd, H.L., Topal, E. and Cardineau, G.A. 2008. P19-dependent and P19-independent reversion of F1-V gene silencing in tomato.Plant Molecular Biology 68:61-79. Angel, C.A., Hsieh, Y.C., Schoelz, J.E. 2011. Comparative analysis of the capacity of tombusvirus P22 and P19 proteins to function as avirulence determinants in Nicotiana species. Molecular Plant-Microbe Interactions 24:91-99. Arnold, M., Durairaj, V., Mundt, E., Schulze, K., Breunig, K.D., Behrens, S.E.2012. Protective vaccination against infectious bursal disease virus with whole recombinant Kluyveromyces lactis yeast expressing the viral VP2 subunit. PLoS ONE 7:e42870. Azad, A. A. Mckern, N. M., Macreadie, I. G., Failla, P., Heine, H. G., Chapman, A., Ward, C. W., Fahey, K. J. 1991. Physicochemical and immunological characterization of recombinant host-protective antigen (VP2) of infectious bursal disease virus. Vaccine 9:715-722. Azhakanandam, K., Weissinger, S.M., Nicholson, J.S., Qu, R. and Weissinger, A.K. 2007. Amplicon-plus targeting technology (APTT) for rapid production of a highly unstable vaccine protein in tabacco plants. Plant Molecular Biology 63:393-404. Baulcombe, D. 2004. RNA silencing in plants. Nature 431:356-363. Berg, T.P. 2000. Acute infectious bursal disease in poultry: a review. Avian pathology. World Veterinary Poultry Association 29:175-194. Birnboim, H.C., and Doly, J. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research 7:1513-1523. Brodersen, P., and Voinnet, O. 2006. The diversity of RNA silencing pathways in plants. Trends in Genetics 22:268–280. Brown, M.D., Skinner, M.A. 1996. Coding sequences of both genome segment of a European 'very virulent' infectious bursal disease virus. Virus Research 40:1-15. Bruchmüller, A., Marthe, C., Hensel, G., Sode, B., Goedeke, S., Borisjuk, N., Brodzik, R., Koprowski, H., Kumlehn, J. 2007. Expression of influenza A (H5N1) vaccine in barley grains for oral bird immunization. Journal Für Verbraucherschutz und Lebensmittelsicherheit 2:118-118. Canizares, M.C., Nicholson, L., Lomonossoff, G. P. 2005. Use of viral vectors for vaccine production in plants. Immunol Cell Biology 83:263-270. Chen, C.S., Suen, S.Y., Chang, G.R., Lu, T.C. Lee, M.S., Wang, M.Y. 2005. Purification of capsid-like particles of infectious bursal disease virus (IBDV) VP2 expressed in E. coli with a metal-ion affinity membrane system. Joural of Virological Methods 130:51-58. Chen, T.H., Chen, T.H., Hu, C.C., Liao, J.T., Lee, C.W., Liao, J.W., Lin, M.Y., Liu, H.J., Wang, M.Y., Lin, N.S., Hsu, Y.H. 2012. Induction of protective immunity in chickens immunized with plant-made chimeric Bamboo mosaic virus particles expressing very virulent Infectious bursal disease virus antigen. Virus Research 166:109-115. Dager, M., and Ehrlich, S.D. 1979. Prolonged incubation in calcium chloride imporoves the competence of Escherichia coli cells. Gene:23-28. Darteil, R., Bublot, M., Laplace, E., Bouquet, J.F., Audonnet, J.C., Riviere, M. 1995. Herpesvirus of turkey recombinant virus expressing infectious bursal diease virus (IBDV) VP2 immunogen induce protection against an IBDV virulent challenge in chickens. Virology 211:481-490. Daniell, H., Singh, N.D., Mason, H., Streatfield, S.J. 2009. Plant-made vaccine antigens and biopharmaceuticals. Trends in Plant Science 14:669-679. Delmas, B., Mundt, E., Vakharia, V.N., Wu, J.L. 2011. Family birnaviridae, in : King, A.M.Q., Lefkowitz, E., Adams, M.J., Carstens, E.B. (Eds.), Virus taxonomy ninth report of the international committee on taxonomy of viruses. academic press, Inc., San Diego, Calif Sharma. p499-507. Fachey, K. J., Katrina, E., Jennifer C. 1989. A conformational immunogen on VP2 of infectious bursal disease virus that induces virus-neutralizing antibodies that passively protect chichens. Journal of General Virology 70:1473-1481. Francois, A., Eterradossi, N., Delmas, B., Payet, V., Langlois, P. 2001. Construction of avian adenovirus CELO recombinant in cosmids. Journal of Virology 75:5288-5301. Franocois, A., Chevalier, C., Delmas, B., Eterradossi, N., Toquin, D., Rivallan, G., Langlois, P. 2004. Avian adenovirus CELO recombinant expressing VP2 of infectious bursal disease vorius induce protection against bursal disease in chickens. Vaccine 22:2351-2360. Garabagi, F., Gilbert, E., Loos, A., McLean, M.D., Hall, J.C. 2012. Utility of the P19 suppressor of gene-silencing protein for production of therapeutic antibodies in Nicotiana expression hosts. Plant Biotechnology Journal 10:1118-1128. Gomez, E., Lucero, M.S., Chimeno Zoth, S., Carballeda, J.M., Gravisaco, M.J., Berinstein, A. 2013. Transient expression of VP2 in Nicotiana benthamiana and its use as a plant-based vaccine against infectious bursal disease virus. Vaccine 31:2623-2627. Goudarzi, H., Seyfiabad Shapouri, M.R., Toroghi, R., Azizy, M., Shahbazzadeh, D., Pourbakhsh, S.A. 2006. Expression of VP2 protein of classical infectious bursal disease virus in the methylotrophic yeast Pichia pastoris. Archives of Razi Institute 61:131-136. Grimsley, N., Hohn, Y., Davies, J. W. and Hohn, B. 1987. Agrobacterium-mediated delivery if infectious maize streak virus into maize plants. Nature 325:177-179. Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. Journal of Molecular Biology 166:557-580. Harwood, W.A., Bartlett, J.G., Alves, S.C., Perry, M., Smedley, M.A., Leyland, N., Snape, J.W. 2009. Barley transformation using Agrobacterium-mediated techniques. Methods in Molecular Biology 478:137-147. Holme, I.B., Brinch-Pedersen, H., Lange, M., Holm, P.B. 2006. Transformation of barley (Hordeum vulgare L.) by Agrobacterium tumefaciens infection of in vitro cultured ovules. Plant Cell Reports 25:1325-1335. Holme, I.B., Brinch-Pedersen, H., Lange, M., Holm, P.B. 2008. Transformation of different barley (Hordeum vulgare L.) cultivars by Agrobacterium tumefaciens infection of in vitro cultured ovules. Plant Cell Reports 27:1833-1840. Holme, I.B., Brinch-Pedersen, H., Lange, M., Holm, P.B. 2012. Transformation of barley (Hordeum vulgare L.) by Agrobacterium tumefaciens infection of in vitro cultured ovules. Methods in Molecular Biology 847:151-161. Horsch, R. B., Fry, J. E., Hoffmann, N. L., Eichholtz, D., Rogers, S. G., Fraley, R. T. 1985. A simple general method for transferring genes into plants. Science 227:1229-1231. Hsieh, M.K., Wu, C.C., Lin, T.L. 2010. DNA-mediated vaccination conferring protection against infectious bursal disease in broiler chickens in the presence of maternal antibody. Vaccine 28:3936-3943. Huang, W.E., Huang, L., Preston, G., Naylor, M., Carr, J.P., Li, Y., Singer, A.C., Whiteley, A.S., Wang, H. 2006. Quantitative in situ assay of salicylic acid in tobacco leaves using a genetically modified biosensor strain of Acinetobacter sp. ADP1. Plant Journal 46:1073-1083. Huang, Z., Elankumaran, S., Yunus, A.S., Samal, S.K. 2004. A recombinant Newcastle disease virus (NDV) expressing VP2 protein of infectious bursal disease virus (IBDV) protects against NDV and IBDV. Journal of Viral 78: 10054-10063. Hudson, P.J., N. M. Mckern, B. E. Power., A. A. Azad. 1986. Genomic structure of the large RNA segment of infectious bursal disease virus. Nucleic Acids Research. 14:5001-5012. Huw D. Jones and Peter R. Shewry. 2006. Methods in molecular biology, transgenic wheat, barley and oats, vol. 478. Hwang, Y.S., Yang, D., McCullar, C., Wu, L., Chen, L., Pham, P., Nandi, S., Huang, N. 2002. Analysis of the rice endosperm-specific globulin promoter in transformed rice cells. Plant Cell Report 20:842-847. Ish-Hormowitz, D. and Burke, J. F. 1981. Rapid and efficient comsid cloning. Nucleic Acids Research 9:2989-2998. Jagadish, M.N., Staton, V.J., Hudson, P, J., Azad, A.A. 1998. Birnavirus precursor polyprotein is processed in Escherichia coli by its own virus-encoded polypeptide. Journal of Virology 62:1084-1087. Jim M. Dunwell and Andy C. Wetten. 2012. Transgenic plants: methods and protocols. Methods in Molecular Biology, vol. 847. Judge, N.A., Mason, H.S., O'Brien, A.D. 2004. Plant cell-based intimin vaccine given orally to mice primed with intimin reduces time of Escherichia coli O157: H7 shedding in feces. Infection and Immunity 72:168–175. Karthikeyan, A., Pandian, S.K., Ramesh, M. 2011. Agrobacterium-mediated transformation of leaf base derived callus tissues of popular indica rice (Oryza sativa L. sub sp. indica cv. ADT 43). Plant science : an International Journal of Experimental Plant Biology 181:258-268. Komarova, T.V., Skulachev, M.V., Zvereva, A.S., Schwartz, A.M., Dorokhov, Y.L., Atabekov, J.G. 2006. New viral vector for efficient production of target proteins in plants. Biochemistry 71:846-850. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. Li, Y.I., Cheng, Y.M., Huang, Y.L., Tsai, C.H., Hsu, Y.H., Meng, M. 1998. Identification and characterization of the Escherichia coli-expressed RNA-dependent RNA polymerase of bamboo mosaic virus. Journal of Virology 72:10093-10099. Li, Y.I., Shih, T.W., Hsu, Y.H., Han, Y.T., Huang, Y.L., Meng, M. 2001. The helicase-like domain of plant potexvirus replicase participates in formation of RNA 5' cap structure by exhibiting RNA 5' triphosphatase activity. Journal of Virology 75:12114-12120. Lin, M.K., Cheng, B.Y., Liao, J.T., Lin, N.S., Hsu, Y.H. 2004. Arg-16 and Arg-21 in the N-terminal region of the triple-gene block protein 1 of bamboo mosaic virus are essential for virus movement. Journal of General Virology 85:251-259. Lin, M.T., Kitajima, E.W., Cupertino, F.P., Costa, C.L. 1977. Partial purification and some properties of bamboo mosaic virus. Phytopathology 67:1433-1439. Lin, N.S., and Hsu, Y.H. 1994. A satellite RNA associated with bamboo mosaic potexvirus. Virology 202:707-714. Lin, N. S., Y. J. Chai, T.Y. Huang, T.Y. Chang., Y.H. Hsu., 1993. Incidence of bamboo mosaic potexvirus in Tiwan. Plant Disease 77:448-450. Lindbo, J.A. 2007. High-efficiency protein expression in plants from agroinfection-compatible Tobacco masaic virus expression vector. BMC Biotechnol 7:52. Lot, H., Marrou, J., Quiot, J. B., Esvan, C. 1972. Contribution a l'etude de virus de la mosaique du concombre (CMV). II. Methode de purification rapide du virus. Phytopathology 4:25–38. Marillonnet, S., Thoeringer, C., Kandzia, R., Klimyuk, V., Gleba, Y. 2005. Systemic Agrobacterium tumefaciens-mediated transfection of viral replicons for efficient transient expression in plants. Nature Biotechnology 23:718-723. Mason, H.S., Lam, D.M.K., Arntzen, C.J. 1992. Expression of hepatitis B surface antigen in transgenic plants. Proc Natl Acad Sci. USA 89:11745–11749. Molina, A., Veramendi, J., Hervas-Stubbs, S. 2005. Induction of neutralizing antibodies by a tobacco chloroplast-derived vaccine base on a B cell epitope from canine parvovirus. Virology 342:266-275. Morgan, M. M., Macreadie, I. G., Harley, V. R., Hudson, P. J., Azad, A.A. 1988. Sequence of the small double-stranded RNA genomic segment of infectious bursal disease virus and its deduced 90-kDa product. Virology 163:240–242. Murashinge, T., and Skfoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Phsiol plant 15:473-497. Park, J.H., Sung, H.W., Yoon, B.I., Kwon, H.M. 2009. Protection of chicken against very virulent IBDV provided by in ovo priming with DNA vaccine and boosting with killed vaccine and the adjuvant effects of plasmid-encoded chicken interleukin-2 and interferon-gamma. Journal of Veterinary Science 10:131-139. Pitcovski, J., Gallili, G., Goldway, M., Perelman, B., Gross, G, Krispel, S., Barbakov, M., Michael, A. 2003. Development and large-scale use of recombinant VP2 vaccine for the prevention of infectious bursal disease of chickens. Vaccine 21:4736-4743. Pradhan, S.N., Prince, P.R., Madhumathi, J., Roy, P., Narayanan, R.B., Antony, U. 2012. Protective immune responses of recombinant VP2 subunit antigen of infectious bursal disease virus in chickens. Veterinary immunology and immunopathology 148:293-301. Pumplin, N., Voinnet, O. 2013. RNA silencing suppression by plant pathogens: defence, counter-defence and counter-counter-defence. Nature reviews. Microbiology 11:745-760. Sambrook, J., Fritsch, E.F., Maniatis, T. 1989. Molecular cloning: a laboratory manual, second edition. Cold spring harbor laboratory press, New York. Saxena, P., Hsieh, Y.C., Alvarado, V.Y., Sainsbury, F., Saunders, K., Lomonossoff, G.P., Scholthof, H.B. 2011. Improved foreign gene expression in plants using a virus-encoded suppressor of RNA silencing modified to be developmentally harmless. Plant Biotechnology 9:703–712. Shaw, I., and Davison, T.F. 2000. Protection from IBDV-induced bursal damage by a recombinant fowlpox vaccine, fpIBD1, is dependent on the titre of challenge virus and chicken genotype. Vaccine 18:3230-3241. Sheppard, M., Werner, W., Tsatas, E., McCoy, R., Prowse, S., Johnson, M. 1998. Fowl adenovirus recombinant expression VP2 of infectious bursal disease virus induces protective immunity against bursal disease. Archives of Virology 143: 915-930. Siddiqui, S.A., Sarmiento, C., Truve, E., Lehto, H., Lehto, K. 2008. Phenotype and functional effects caused by various viral RNA silencing suppressors in transgenic Nicotiana benthamiana and N. tabacum. Molecular plant-Microbe Interactions 21:178–187. Spies U, and Müller H. 1990. Demonstration of enzyme activities required for cap structure formation in infectious bursal disease virus, a member of the birnavirus group. Journal of General Virology 71:977–981. Spies, U., Müller, H., Becht, H. 1987. Properties of RNA polymerase activity associated with infectious bursal disease virus and characterization of its reaction products. Virus Research 8:127-140. Takagi, H., Saito, S., Yang, L., Nagasaka, S., Nishizawa, N., Takaiwa, F. 2005. Oral immunotherapy against a pollen allergy using a seed-base peptide vaccine. Plant Biotechnology 3:521–533. Takaiwa, F., Yamanouchi, U., Yoshihara, T., Washida, H., Tanabe, F., Kato, A., Yamada, K. 1996. Characterization of common cis- regulatory elements responsible for the endosperm-specific expression of members of the rice glutelin multigene family. Plant molecular Biology 30:1207–1221. Tsukamoto, K., Sato, T., Saito, S., Tanimura, N., Hamazaki, N., Mase, M., Yamaguchi, S. 2000. Dual-virus vector approach induced strong and long-lasting protective immunity against very vitulent-infectious bursal disease virus. Virology 269:257-267. Tsukamoto, K., Sato, S., Saeki, S., Sato, T., Tanimura, N., Isobe, T., Mase, M., Imada, T., Yuasa, N., Yamaguchi, S., 2002. Complete, long-lasting protection against lethal infectious bursal disease virus challenge by a single vaccination with an avian herpesvirus vector expressing VP2 antigens. Journal of Virology 76:5637-5645. Vakharia, V.N., Snyder, D.B., Lutticken, D., Mengel-Whereat, S.A., Savage, P.K., Edwards, G.H., Goodwin, M.A. 1994. Active and passive protection against variant and classic infectious bursal disease virus strains induced by baculovirus-expressed structural proteins. Vaccine 12:452-456. Verwoerd, T.C., Dekker, B.M.M., Hoekema, A. 1989. A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Research 17:2362. Voinnet, O., Rivas, S., Mestre, P., Baulcombe, D. 2003. An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant of Journal 33:949–956. Wang, M. Y., Doong, S. R. 2000. A pH-based fed-batch process for the production of a chimeric recombinant infectious bursal disease (IBDV) structural protein (rVP2H) in insect cells. Process Biochemistry 35:877-884. Wang, M.Y., Kup, Y.Y., Lee, M.S., Doong, S.R., Ho, J.Y., Lee, L.H. 2000. Sele-assembly of the infectious bursal disease virus capsid protein, rVP2, expressed in insect cells and purification of immunogenic chimeric rVP2H particles by immobilized metal-ion affinity chromatography. Biotechnology and Bioengineering 67:104-111. Warzecha, H., Mason, H.S. 2003. Benefits and risks of antibody and vaccine production in transgenic plants. Journal of Plant Physiology 160:755-764. Wu, C., Suzuki, A., Washida, H. and Takaiwa, F. 1998. The GCN4 motif in a rice glutelin gene is essential for endosperm-specific gene expression and is activated by Opaque-2 in transgenic rice plants. Plant Journal 14:1361–1364. Wu, H., Singh, N.K., Locy, R.D., Scissum-Gunn, K., Giambrone, J.J. 2004. Immunization of chickens with VP2 protein of infectious bursal disease virus expressed in Arabidopsis thaliana. Avian Diseases 48:663-668. Wu, J., Yu, L., Li., Hu, J., Zhou, J., Zhou, X. 2007. Oral immunization with transgenic rice seeds expressing VP2 protein of infectious bursal disease virus induces protective immune responses in chickens. Plant Biotechnology Journal 5: 570-578. Wung, C.H., Hsu, Y.H., Liou, D.Y., Huang, W.C., Lin, N.S., Chang, B.Y. 1999. Identification of the RNA-binding sites of the triple gene block protein 1 of bamboo mosaic potexvirus. Journal of General Virology 80:1119-1126. Xu, X.G., Tong, D.W., Wang, Z.S., Zhang, Q., Li, Z.C. 2011. Baculovirus virions displaying infectious bursal disease virus VP2 protein protect chickens against infectious bursal disease virus infection. Avian Disease 55:223-229. Yang, C.C., Liu, J.S., Lin, C.P., Lin, N.S. 1997. Nucleotide sequence and phylogenetic analysis of bamboo mosaic potexvirus isolate from common bamboo (Bambusa vulgaris McClure). Bot. Bull. Acad. Sin 38:77-84. Yang, C.D., Liao, J.T., Lai, C.Y., Jong, M.H., Liang, C.M., Liang Y., Lin, N.S., Hsu, Y.H., Liang, S.M. 2007. Induction of protective immunity in swin by recombinant bamboo mosaic virus expression foot-and-mouth disease virus epitopes. BMC Biotechnology 7:62. Yang, D., Guo, F., Liu, B., Huang, N. and Watkins, S.C. 2003. Expression and localization of human lysozyme in the endosperm of transgenic rice. Planta 216:597-603. Yao, K., Vakharia, V.N. 2001. Induction of apoptosis in vitro by the 17-kDa nonstructural protein of infectious bursal disease virus: possible role in viral pathogenesis. Virology 285:50-58. Yeh, C.C., Cheng, A.H., Hwang, H.Y. 1993. Indexing of bamboo mosaic virus and propagation of virus-free bamboo plants. Proceedings of Symposium on Plant Virus and Virus-like Disease. Plant Protection 1:275-282. Zheng, N., Xia, R., Yang, C., Yin, B., Li, Y., Duan, C., Liang, L., Guo, H., Xie, Q. 2009. Boosted expression of the SARS-CoV nucleocapsid protein in tobacco and its immunogenicity in mice. Vaccine 27:5001–5007. Zhou, X., Wang, D., Xiong, J., Zhang, P., Li, Y., She, R. 2010. Protection of chickens, with or without maternal antibodies, against IBDV infection by a recombinant IBDV-VP2 protein. Vaccine 28:3990-33996.
摘要: 
傳染性華氏囊病毒(Infectious bursal disease virus, IBDV)是一種急性且具高傳染性的病毒,為引起雞華氏囊病的主因,主要發生在 3-6 週的幼雛,常造成雞隻養殖場嚴重的經濟損失,因此疫苗的使用為當務之急。目前在此疫苗的發展上,除了一般的不活化病毒疫苗或減毒活疫苗之外,近幾年也有學者致力研究以植物做為生產平台,藉此以生產安全、方便且價格低廉之植物生產疫苗。因此,本研究分別使用 Bamboo mosaic virus (BaMV)、Tobacco mosaic virus(TMV)、Foxtail mosaic virus (FoMV)及 Potato virus X (PVX) 四種植物病毒做為載體,利用華氏囊病毒 VP2 基因取代病毒鞘蛋白,並於其 C 端融合 His-tag 形成功能缺失性的重組病 毒載體 BaMVdC-VP2His 、 TMVdC-VP2His 、FoMVdC-VP2His 及 PVXdC-VP2His,以農桿菌接種至菸草植物葉片上偵測 VP2His蛋白表現,結果顯示 4 種載體中以 BaMV 載體之 VP2His 蛋白表現量為最高。接著以菸草(Nicotiana benthamiana) 為材料 , 利用農桿菌轉殖方式建立BaMVdC-VP2His 轉殖植物,經西方墨點轉漬法篩選得到外表正常又能穩定表現蛋白的同源植物 (homologous line);由轉殖植物中純化 VP2 蛋白,於穿透式電子顯微鏡下可觀察到直徑大約為 20-30 nm 的 VP2 類病毒顆粒。為了顧及環境安全性和獲得更高的外源蛋白 以不同基因靜默抑制子(gene silencing suppressor),取代病毒三重疊基因區(triple gene block, TGB),利用基因靜默子和 BaMV 載體共同表現為策略,和嘗試共同表現竹嵌紋病毒鞘蛋白,結果顯示外源蛋白表現量並無明顯增加。為了研發食用疫苗,將 BaMVdC-VP2His 重組病毒基因體轉殖到大麥(Hordeum vulgare L.)中,雖然在大麥上的表現量不如預期,但已建立大麥轉殖的系統。未來由轉殖植物中純化出 VP2 類病毒顆粒進行動物試驗,偵測疫苗的中和抗性及效價,並以此系統發展次單位疫苗對抗雞華氏囊病毒。

Infectious bursal disease virus (IBDV) is an acute and highly contagious virus which is main cause of infectious bursal disease. It primarily occurrs in 3-6 weeks chickens and seriously affects the economy loss of poultry industry. Currently, inactivated and attenuated vaccines are two main strategies for chicken IBD prevention. In recent years, the safer and low-cost vaccines are produced by using plants as a platform. In this study, we constructed Bamboo mosaic virus (BaMV), Tobacco mosaic virus (TMV), Foxtail mosaic virus (FoMV), and Potato virus X (PVX) as plant viral vectors. The coat protein genes of viral vectors are replaced with IBDV VP2 gene and fused with His-tag in the C-terminal, to generate BaMVdC-VP2His, TMVdC-VP2His, FoMVdC-VP2His, and PVXdC-VP2His. To test the VP2 production, these four recombinant viruses were individually transfected into Nicotiana benthamiana leaves by agro-infiltration. The results showed that among four viral vectors, BaMV vector expressed the most high-level accumulation of VP2 protein. In order to stably express VP2 protein in plants, we generated BaMVdC-VP2His transgenic N. benthamiana plants by Agrobacterium- mediated transformation. Western blot analysis confirmed the VP2 protein could be expressed in homologous transgenic lines. The virus like particles (VLP) of VP2 approximately 20-30 nm in diameter were purified and observed by transmission electron microscopy. Taking into account the environmental safety and high expression of VP2His protein, different gene silencing suppressors replace BaMV TGB or co-expression with BaMVdC-VP2His. Furthermore, complementation coat protein function of BaMVdC-VP2His by co-expression of BaMV CP. The results indicate VP2 protein dose not significantly increase. To develop edible vaccine, the transgenic barley (Hordeum vulgare) were generated. The results showed that BaMVdC-VP2His transgenic barley accumulated low VP2 protein. However, we have successfully established tissue culture and plant regeneration system in barley. We will use VP2-VLP to immuning chicken and to test the neutralizing antibodies production. We hope in the future, to develop subunit vaccine against IBDV by the BaMV based vector system.
URI: http://hdl.handle.net/11455/90043
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