Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/66427
標題: 假性狂犬病毒醣蛋白gE的表現及應用
Expression and application of the pseudorabies virus (PRV) glycoprotein E (gE)
作者: 楊于萱
Yang, Yu-Shiuan
關鍵字: PRV;假性狂犬病毒;glycoprotein E;monoclonal antibody;醣蛋白gE;單源抗體
出版社: 獸醫微生物學研究所
引用: Ao, J.Q., Wang, J.W., Chen, X.H., Wang, X.Z. & Long, Q.X. (2003) Expression of pseudorabies virus gE epitopes in Pichia pastoris and its utilization in an indirect PRV gE-ELISA. Journal of virological methods, 114, 145-150. Brideau, A.D., Banfield, B.W. & Enquist, L.W. (1998) The Us9 gene product of pseudorabies virus, an alphaherpesvirus, is a phosphorylated, tail-anchored type II membrane protein. Journal of virology, 72, 4560-4570. Brideau, A.D., Eldridge, M.G. & Enquist, L.W. (2000) Directional transneuronal infection by pseudorabies virus is dependent on an acidic internalization motif in the Us9 cytoplasmic tail. Journal of virology, 74, 4549-4561. Buchacher, A., Predl, R., Strutzenberger, K., Steinfellner, W., Trkola, A., Purtscher, M., Gruber, G., Tauer, C., Steindl, F., Jungbauer, A. & et al. (1994) Generation of human monoclonal antibodies against HIV-1 proteins; electrofusion and Epstein-Barr virus transformation for peripheral blood lymphocyte immortalization. AIDS Res Hum Retroviruses, 10, 359-369. Card, J.P., Whealy, M.E., Robbins, A.K. & Enquist, L.W. (1992) Pseudorabies virus envelope glycoprotein gI influences both neurotropism and virulence during infection of the rat visual system. Journal of virology, 66, 3032-3041. Ch''ng, T.H. & Enquist, L.W. (2005) Efficient axonal localization of alphaherpesvirus structural proteins in cultured sympathetic neurons requires viral glycoprotein E. Journal of virology, 79, 8835-8846. Chang, Y.Y., Lin, H.W., Wong, M.L. & Chang, T.J. (2004) Regulation of the vhs gene promoter of pseudorabies virus by IE180 and EP0, and the requirement of a Sp1 Site for the promoter function. Virus Genes, 28, 247-258. Chang, Y.Y., Wong, M.L., Lin, H.W. & Chang, T.J. (2002) Cloning and regulation of the promoter of pseudorabies virus (TNL strain) glycoprotein E gene. Virus Genes, 24, 235-241. Chowdhury, S.I., Mahmood, S., Simon, J., Al-Mubarak, A. & Zhou, Y. (2006) The Us9 gene of bovine herpesvirus 1 (BHV-1) effectively complements a Us9-null strain of BHV-5 for anterograde transport, neurovirulence, and neuroinvasiveness in a rabbit model. Journal of virology, 80, 4396-4405. Collins, W.J. & Johnson, D.C. (2003) Herpes simplex virus gE/gI expressed in epithelial cells interferes with cell-to-cell spread. Journal of virology, 77, 2686-2695. de StGroth, S.F. & Scheidegger, D. (1980) Production of monoclonal antibodies: strategy and tactics. J Immunol Methods, 35, 1-21. Donald Voet, J.G.V., Charlotte W. Pratt (2000) Fundamentals of Biochemistry. John Wiley & Sons, Inc., New York. Ehrlich, P.H., Moyle, W.R. & Moustafa, Z.A. (1983) Further characterization of cooperative interactions of monoclonal antibodies. J Immunol, 131, 1906-1912. Enquist, L.W. (1999) Life beyond eradication: veterinary viruses in basic science. Arch Virol Suppl, 15, 87-109. Enquist, L.W., Husak, P.J., Banfield, B.W. & Smith, G.A. (1998) Infection and spread of alphaherpesviruses in the nervous system. Adv Virus Res, 51, 237-347. Estes, D.M., Closser, N.M. & Allen, G.K. (1994) IFN-gamma stimulates IgG2 production from bovine B cells costimulated with anti-mu and mitogen. Cell Immunol, 154, 287-295. Farnsworth, A. & Johnson, D.C. (2006) Herpes simplex virus gE/gI must accumulate in the trans-Golgi network at early times and then redistribute to cell junctions to promote cell-cell spread. Journal of virology, 80, 3167-3179. Feldman, L., Rixon, F.J., Jean, J.H., Ben-Porat, T. & Kaplan, A.S. (1979) Transcription of the genome of pseudorabies virus (A herpesvirus) is strictly controlled. Virology, 97, 316-327. Ficinska, J., Van Minnebruggen, G., Nauwynck, H.J., Bienkowska-Szewczyk, K. & Favoreel, H.W. (2005) Pseudorabies virus glycoprotein gD contains a functional endocytosis motif that acts in concert with an endocytosis motif in gB to drive internalization of antibody-antigen complexes from the surface of infected monocytes. Journal of virology, 79, 7248-7254. Fuchs, W., Klupp, B.G., Granzow, H., Hengartner, C., Brack, A., Mundt, A., Enquist, L.W. & Mettenleiter, T.C. (2002) Physical interaction between envelope glycoproteins E and M of pseudorabies virus and the major tegument protein UL49. Journal of virology, 76, 8208-8217. Galfre, G. & Milstein, C. (1981) Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol, 73, 3-46. Henis, Y.I., Herman-Barhom, Y., Aroeti, B. & Gutman, O. (1989) Lateral mobility of both envelope proteins (F and HN) of Sendai virus in the cell membrane is essential for cell-cell fusion. J Biol Chem, 264, 17119-17125. Honess, R.W. & Roizman, B. (1974) Regulation of herpesvirus macromolecular synthesis. I. Cascade regulation of the synthesis of three groups of viral proteins. Journal of virology, 14, 8-19. Jacobs, L. (1994) Glycoprotein E of pseudorabies virus and homologous proteins in other alphaherpesvirinae. Arch Virol, 137, 209-228. Jacobs, L., Meloen, R.H., Gielkens, A.L. & Van Oirschot, J.T. (1990) Epitope analysis of glycoprotein I of pseudorabies virus. J Gen Virol, 71 ( Pt 4), 881-887. Kaplan, A.S. & Vatter, A.E. (1959) A comparison of herpes simplex and pseudorabies viruses. Virology, 7, 394-407. Kimman, T.G., de Leeuw, O., Kochan, G., Szewczyk, B., van Rooij, E., Jacobs, L., Kramps, J.A. & Peeters, B. (1996) An indirect double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) using baculovirus-expressed antigen for the detection of antibodies to glycoprotein E of pseudorabies virus and comparison of the method with blocking ELISAs. Clin Diagn Lab Immunol, 3, 167-174. Klock, G., Wisnewski, A.V., el-Bassiouni, E.A., Ramadan, M.I., Gessner, P., Zimmermann, U. & Kresina, T.F. (1992) Human hybridoma generation by hypo-osmolar electrofusion: characterization of human monoclonal antibodies to Schistosoma mansoni parasite antigens. Hybridoma, 11, 469-481. Klupp, B.G., Altenschmidt, J., Granzow, H., Fuchs, W. & Mettenleiter, T.C. (2005) Identification and characterization of the pseudorabies virus UL43 protein. Virology, 334, 224-233. Klupp, B.G., Hengartner, C.J., Mettenleiter, T.C. & Enquist, L.W. (2004) Complete, annotated sequence of the pseudorabies virus genome. Journal of virology, 78, 424-440. Knapp, A.C., Husak, P.J. & Enquist, L.W. (1997) The gE and gI homologs from two alphaherpesviruses have conserved and divergent neuroinvasive properties. Journal of virology, 71, 5820-5827. Kohler, G. & Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 256, 495-497. Kohler, M. & Kohler, W. (2003) Zentralblatt fur Bakteriologie--100 years ago Aladar aujeszky detects a ''new'' disease--or: it was the cow and not the sow. Int J Med Microbiol, 292, 423-427. Kritas, S.K., Pensaert, M.B. & Mettenleiter, T.C. (1994) Role of envelope glycoproteins gI, gp63 and gIII in the invasion and spread of Aujeszky''s disease virus in the olfactory nervous pathway of the pig. J Gen Virol, 75 ( Pt 9), 2319-2327. Lewin, B. (2004) Genes VIII. Pearson Prentice Hall Pearsopn Education, Inc., United States of America. Littlefield, J.W. (1964) Selection of Hybrids from Matings of Fibroblasts in Vitro and Their Presumed Recombinants. Science, 145, 709-710. Lokensgard, J.R., Thawley, D.G. & Molitor, T.W. (1990) Pseudorabies virus latency: restricted transcription. Arch Virol, 110, 129-136. Maes, R.K., Sussman, M.D., Vilnis, A. & Thacker, B.J. (1997) Recent developments in latency and recombination of Aujeszky''s disease (pseudorabies) virus. Vet Microbiol, 55, 13-27. Martinez-Torrecuadrada, J.L. & Casal, J.I. (1995) Identification of a linear neutralization domain in the protein VP2 of African horse sickness virus. Virology, 210, 391-399. McGeoch, D.J. & Cook, S. (1994) Molecular phylogeny of the alphaherpesvirinae subfamily and a proposed evolutionary timescale. J Mol Biol, 238, 9-22. Mettenleiter, T.C. (2000) Aujeszky''s disease (pseudorabies) virus: the virus and molecular pathogenesis--state of the art, June 1999. Vet Res, 31, 99-115. Mettenleiter, T.C. (2002) Brief overview on cellular virus receptors. Virus Res, 82, 3-8. Mettenleiter, T.C. (2006) Intriguing interplay between viral proteins during herpesvirus assembly or: the herpesvirus assembly puzzle. Vet Microbiol, 113, 163-169. Mettenleiter, T.C., Zsak, L., Kaplan, A.S., Ben-Porat, T. & Lomniczi, B. (1987) Role of a structural glycoprotein of pseudorabies in virus virulence. Journal of virology, 61, 4030-4032. Nauwynck, H., Glorieux, S., Favoreel, H. & Pensaert, M. (2007) Cell biological and molecular characteristics of pseudorabies virus infections in cell cultures and in pigs with emphasis on the respiratory tract. Vet Res, 38, 229-241. Newby, T.J., Carter, D.P., Yoon, K.J., Jackwood, M.W. & Hawkins, P.A. (2002) Assessment of replication and virulence of attenuated pseudorabies virus in swine. J Vet Sci, 3, 61-66. Ou, C.J., Wong, M.L. & Chang, T.J. (2002) A TEF-1-element is required for activation of the promoter of pseudorabies virus glycoprotein X gene by IE180. Virus Genes, 25, 241-253. Peeters, B., Pol, J., Gielkens, A. & Moormann, R. (1993) Envelope glycoprotein gp50 of pseudorabies virus is essential for virus entry but is not required for viral spread in mice. Journal of virology, 67, 170-177. Pensaert, M.B., and J. P. Kluge. (1989) "Pseudorabies virus (Aujesky''s disease)" virus infections of procines. Elsevier Science Publishers, B. V. Amsterdam. Petrovskis, E.A., Timmins, J.G. & Post, L.E. (1986) Use of lambda gt11 to isolate genes for two pseudorabies virus glycoproteins with homology to herpes simplex virus and varicella-zoster virus glycoproteins. Journal of virology, 60, 185-193. Pomeranz, L.E., Reynolds, A.E. & Hengartner, C.J. (2005) Molecular biology of pseudorabies virus: impact on neurovirology and veterinary medicine. Microbiol Mol Biol Rev, 69, 462-500. Quint, W., Gielkens, A., Van Oirschot, J., Berns, A. & Cuypers, H.T. (1987) Construction and characterization of deletion mutants of pseudorabies virus: a new generation of ''live'' vaccines. J Gen Virol, 68 ( Pt 2), 523-534. Rziha, H.J., Mettenleiter, T.C., Ohlinger, V. & Wittmann, G. (1986) Herpesvirus (pseudorabies virus) latency in swine: occurrence and physical state of viral DNA in neural tissues. Virology, 155, 600-613. Smith, G.A. & Enquist, L.W. (2002) Break ins and break outs: viral interactions with the cytoskeleton of Mammalian cells. Annu Rev Cell Dev Biol, 18, 135-161. Takahashi, K., Tereda, S., Ueda, H., Makishima, F. & Suzuki, E. (1994) Growth rate suppression of cultured mammalian cells enhances protein productivity. Cytotechnology, 15, 57-64. Tirabassi, R.S. & Enquist, L.W. (1999) Mutation of the YXXL endocytosis motif in the cytoplasmic tail of pseudorabies virus gE. Journal of virology, 73, 2717-2728. Tirabassi, R.S. & Enquist, L.W. (2000) Role of the pseudorabies virus gI cytoplasmic domain in neuroinvasion, virulence, and posttranslational N-linked glycosylation. Journal of virology, 74, 3505-3516. Van Oirschot, J.T. (1987) Intranasal vaccination of pigs against Aujeszky''s disease: comparison with one or two doses of attenuated vaccines in pigs with high maternal antibody titres. Res Vet Sci, 42, 12-16. van Oirschot, J.T., Gielkens, A.L., Moormann, R.J. & Berns, A.J. (1990) Marker vaccines, virus protein-specific antibody assays and the control of Aujeszky''s disease. Vet Microbiol, 23, 85-101. van Oirschot, J.T., Kaashoek, M.J., Rijsewijk, F.A. & Stegeman, J.A. (1996) The use of marker vaccines in eradication of herpesviruses. J Biotechnol, 44, 75-81. van Oirschot, J.T., Rziha, H.J., Moonen, P.J., Pol, J.M. & van Zaane, D. (1986) Differentiation of serum antibodies from pigs vaccinated or infected with Aujeszky''s disease virus by a competitive enzyme immunoassay. J Gen Virol, 67 ( Pt 6), 1179-1182. Vannier, P., Hutet, E., Bourgueil, E. & Cariolet, R. (1991) Level of virulent virus excreted by infected pigs previously vaccinated with different glycoprotein deleted Aujeszky''s disease vaccines. Vet Microbiol, 29, 213-223. Visser, N., and D. Lu‥tticken. 1989. Experiences with a gI-/TK-modified live, pseudorabies virus vaccine: strain begonia, p.I.J.T.v.O., (ed.), V.a.c.o.A.s.d.K.A. & Publishers, D., The Netherlands. Whealy, M.E., Card, J.P., Robbins, A.K., Dubin, J.R., Rziha, H.J. & Enquist, L.W. (1993) Specific pseudorabies virus infection of the rat visual system requires both gI and gp63 glycoproteins. Journal of virology, 67, 3786-3797. Wittmann, G. (1991) Spread and control of Aujeszky''s disease (AD). Comp Immunol Microbiol Infect Dis, 14, 165-173. Wozniak, M.A., Shipley, S.J., Combrinck, M., Wilcock, G.K. & Itzhaki, R.F. (2005) Productive herpes simplex virus in brain of elderly normal subjects and Alzheimer''s disease patients. J Med Virol, 75, 300-306. Xavier Messeguer, R.E., Domènec Farré, Oscar Nuñez, Javier Martínez, M.Mar Albà. (2002) PROMO: detection of known transcription regulatory elements using species-tailored searches. Bioinformatics, 18, 333-334. Yong, T., Huan-chun, C., Shao-bo, X., Ya-li, Q., Qi-gai, H. & Yu-qi, R. (2005) Development of a latex agglutination test using the major epitope domain of glycoprotein E of pseudorabies virus expressed in E. coli to differentiate between immune responses in pigs naturally infected or vaccinated with pseudorabies virus. Vet Res Commun, 29, 487-497. Zuckermann, F.A., Mettenleiter, T.C., Schreurs, C., Sugg, N. & Ben-Porat, T. (1988) Complex between glycoproteins gI and gp63 of pseudorabies virus: its effect on virus replication. Journal of virology, 62, 4622-4626. Zuckermann, F.A., Zsak, L., Mettenleiter, T.C. & Ben-Porat, T. (1990) Pseudorabies virus glycoprotein gIII is a major target antigen for murine and swine virus-specific cytotoxic T lymphocytes. Journal of virology, 64, 802-812. 林孫權、董明澄、劉正義、張照夫、黃萬居、鄭清木。1972。假性狂犬病之發生報告。中華民國微生物學會雜誌 5:56-68。 黃雅如。2005。假性狂犬病毒早期蛋白UL54之功能區分析。中興大學獸醫微生物研究所碩士論文。
摘要: 
假性狂犬病毒 (pseudorabies virus, PRV) 屬於a- 疱疹病毒亞科
(Alphaherpesvirinae),是造成假性狂犬病的病原。豬是PRV 的自然宿主,感
染PRV 會造成仔豬高致死率,以及懷孕母豬的流產或死產,其盛行率遍及全
球,因此導致經濟上重大的損失。疫苗接種是目前控制假性狂犬病的主要方
式,gE 缺損之疫苗已廣泛使用,其優點為可測定豬隻血清中是否具有抗gE
的抗體,來區分受感染及已免疫之動物,因此gE 具有發展成為快速診斷工具
之潛力。本研究即針對gE 基因進行選殖,將PRV TNL 株gE 基因大小為804
bp 亦即表現N 端268 個胺基酸的基因片段,轉殖於pET28 表現載體中,並於
E. coli 中進行大量表現。所得之gE 重組蛋白 (gEN268) 經由SDS-PAGE 及
Western blot 分析之結果顯示,於45 kDa 處可被抗PRV 豬陽性血清所辨識,
確認其具有正確抗原性。將gEN268 做為抗原並免疫BALB/c 小鼠,利用融合
瘤技術進而製備單源抗體。融合瘤細胞以間接免疫螢光染色法 (indirect
immunofluorescence assay, IFA) 進行篩選,並進一步進行單株化。單株化後之
融合瘤細胞以IFA 進行篩選,並以Western blot 分析單源抗體之特異性,證實
所製備之單源抗體能專一性辨認PRV gE 蛋白。此外,本研究針亦對gE 上游
基因特定片段進行選殖,並分析具啟動子活性之功能區。將gE 基因上游不同
DNA 片段轉殖於CMV 啟動子缺損的重組報告載體 (pEGFPDCMV) 中,分別
構築出pEGFP/gEp166、 pEGFP/gEp425 及 pEGFP/gEp1200 等三個重組質
體,將各重組報告質體分別轉染至PK-15 細胞中進行短暫表現,於轉染後48
小時以倒立螢光顯微鏡觀察細胞是否能表現綠色螢光蛋白 (EGFP) 而呈現綠
色螢光。結果顯示gE 基因上游1 到166 核苷酸序列即具有有效的啟動子功能
活性。

Pseudorabies virus (PRV), an alphaherpesvius, is the causative agent of pseudorabies in swine. The pig is the natural host of PRV, which is characterized by a fatal infection in piglets and abortion in pregnant sows. Pseudorabies is an economically important swine disease worldwide, and vaccination is now used widely in the control of this disease. The gE deleted PRV vaccine strain has been developed, which provides an important advantage to distinguish infected animals from vaccinated ones. Thus, PRV glycoprotein E (gE) has been recognized as a suitable diagnostic reagent for pseudorabies. In order to produce gE protein in a large amount, a 804 bp DNA fragment encoding gE epitopes of PRV TNL strain was cloned in E. coli pET28a expression vector. SDS-PAGE and Western blotting assay showed that the fusion protein (gEN268) was approximately 45 kDa and could be recognized by the swine anti-PRV serum. In addition, the gEN268 protein was used as an antigen to immunize BALB/c mice for developing monoclonal antibody specific to PRV gE by hybridoma techniques. The hybridomas secreting specific antibody against PRV gE were characterized by indirect immunofluorescence assay and Western blotting assay. Furthermore, in order to study the promoter function of gE gene, several defined portions of the upstream of gE gene start codon replaced the CMV promoter of pEGFP-N3 reporter vector and three recombinant plasmids pEGFP/gEp166, pEGFP/gEp425 and pEGFP/gEp1200 were generated. The expression of enhanced green fluorescence protein (EGFP) driven by the gE promoter in transfected PK-15 cells was visualized with a fluorescence microscope at the 48 h post-transfection. The results indicated that the region of nucleotide residues 1-166 upstream of gE start codon possessed sufficient promoter activity for expression of PRV gE.
URI: http://hdl.handle.net/11455/66427
其他識別: U0005-0807200818422100
Appears in Collections:微生物暨公共衛生學研究所

Show full item record
 

Google ScholarTM

Check


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.