成功大學博碩士論文系統

簡易檢索 / 詳目顯示

回結果列表

研究生：	唐中道 Tang, Chung-Tao
論文名稱：	豬繁殖與呼吸道症候群病毒之疫苗研發 Development of porcine reproductive and respiratory syndrome vaccines
指導教授：	蕭璦莉 Shiau, Ai-Li
學位類別：	碩士 Master
系所名稱：	醫學院 - 微生物及免疫學研究所 Department of Microbiology & Immunology
論文出版年：	2003
畢業學年度：	91
語文別：	中文
論文頁數：	48
中文關鍵詞：	疫苗、豬繁殖與呼吸道症候群、豬繁殖與呼吸道症候群病毒
外文關鍵詞：	porcine reproductive and respiratory syndrome, PRRSV, PRRS, vaccine
相關次數：	點閱：79 下載：3
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

豬繁殖與呼吸道症候群(porcine reproductive and respiratory syndrome, PRRS)，是目前世界上廣泛的豬隻傳染疾病，在1987年首次被美國報導出來，其病原體PRRS病毒(PRRSV) 為一種具有外套膜的正股RNA病毒，對於懷孕母豬造成生殖道上的缺陷，以及透過感染豬隻鼻黏膜的上皮細胞以及肺部的巨噬細胞、單核球細胞，使得豬隻的免疫系統受到破壞，造成豬隻免疫力的下降，易於遭受二次感染，而導致死亡。由於現今缺乏良好的疫苗，豬繁殖與呼吸道症候群對於我國的養豬事業造成嚴重的衝擊。因此，本研究希望能直接針對台灣本土的豬繁殖與呼吸道症候群病毒發展出更有效的疫苗。
由先前的研究中指出，PRRSV的GP4、GP5、N基因為動物體免疫系統產生中和抗體的目標部位，以及M基因則是誘發細胞性免疫反應的重要抗原位。所以，我們首先將台灣本土分離株PRRSV-KB3，利用反轉錄聚合脢連鎖反應的方法將病毒的GP4、5、6、7四段基因選殖出來，再利用實驗室中已建立的假性狂犬病病毒(pseudorabies virus, PRV)載體，經由連續的藥物篩選，可將選殖的基因插入假性狂犬病病毒gD基因的位置。最後，可產生帶有PRRSV基因的重組假性狂犬病病毒，命名為PrV-CT4、5、6、7。由於重組病毒缺損gD基因，因此只能造成一次的感染，無法再製造出具感染力的病毒後代，提高了重組病毒疫苗的安全性。在小鼠的實驗中，我們發現此重組病毒疫苗的確能引起對抗PRRSV的抗體免疫反應。在未來，希望此疫苗對於國內豬隻的傳染病防治能有所助益。

Porcine reproductive and respiratory syndrome (PRRS), characterized by reproductive failure in sows and respiratory illness in young pigs, is a worldwide porcine disease. It was first reported in 1987 in USA and described as mystery swine disease. The causative agent, porcine reproductive and respiratory syndrome virus (PRRSV), is an enveloped positive-stranded RNA virus. It can infect pigs through nasal epithelial, pulmonary macrophages and monocytes, and cause immunosuppression. Thus, PRRSV is considered to be a major problem to the pork industry. Several PRRS vaccines are available; however, these vaccines confer protection against clinical disease, but do not prevent viral infection. Furthermore, since PRRSV is genetically and antigenically heterogeneous, it is worthwhile to develop new PRRS vaccines based on Taiwan local strains. The aim of this study was to develop pseudorabies virus (PrV) vectors carrying PRRSV genes as vaccines. Therefore, we cloned the ORFs 4, 5, 6 and 7 of PRRSV that encode virion-associated protein (GP4), envelope (GP5), membrane protein (M) and nucleocapsid protein (N), respectively, by RT-PCR. GP4, GP5 and N proteins can produce neutralizing antibodies directed against the virus. In addition, M protein can induce cell-mediated immunity. Therefore, these genes are good targets and important to induce protective immune responses. Using an acycolvir selection system to generate recombinant pseudorabies virus (PrV), the cloned PRRSV genes were inserted in place of the HSV-TK gene of a gE/gD-negative PrV, resulting in the generation of PrV-CT4, 5, 6 and 7. By serial acyclovir selection, the parent virus was killed and recombinant PrV vectors were produced. Because the recombinant PrV vectors lack gD gene, they could not generate infectious progeny, and thus increase the safety. After immunization of the PrV-CT4, 5 and 7, mice could induce anti-PRRSV neutralizing antibody against viral infection. In conclusion, the recombinant PrV is an adequate vector to express and present antigens of PRRSV. These recombinant viruses may be used as vaccines against PRRSV in the future.

Contents
Abstract…………………………………………………………………………..	I
Chinese Abstract…………………………………………………………………	II
Acknowledgement……………………………………………………………….	III
Contents……….…………………………………………………………………	IV
Figure contents…………………………….……………………………………..	VI
Table contents…………………………………………………………………….	VII
Appendix…………………………………………………………………………	VIII
Abbreviation………………………….…………………………………………..	IX
1. Introduction	
1.1 Porcine reproductive and respiratory syndrome virus …...…………….	1
1.2 Development of PRRS vaccine……………...…………………………	2
1.3 Development of Pseudorabies virus as a vaccine vector……...………..	2
1.4 The strategy to produce PRRS vaccines and induction of immunity against PRRSV………………………………………………………… 	5
2. Materials and methods	
2.1 Cells and viruses…………………………………………………....…..	6
2.2 Cloning of the cDNAs of PRRSV-ORF4, 5, 6 and 7...…..…..………...	6
2.3 Construction of pTCY/KB3-orf4, 5, 6 and 7 vectors…….…….……….	7
2.4 Transient expression of PRRSV proteins………………………………	8
2.5 Constructions of pDelta-gD/KB3-orf4, 5, 6 and 7 shuttle vectors..……	8
2.6 Productions of gD-/gE-/Tk-/PRRSV genes+ PrV…..…………………...	9
2.7 Isolation of Viral DNA………………..…………………….………….	9
2.8 PCR detection of PrV-CT4, 5, 6 and 7…………………………..……..	10
2.9 Transgene expression in Vero cells infected with PrV-CT……..………	11
2.10 Determination of TCID50 of PrV-CT.….……………………………...	11
2.11 Immunization of mice with PrV-CT4, 5, 6 and 7….………………….	12
2.12 Detection of anti-PRRSV and PrV antibody by ELISA………………	12
2.13 Detection of anti-PRRSV neutralizing antibody by neutralizationantibody
     (NT) test…        …………………………………………………..	13
2.14 Detection of anti-PrV neutralizing antibody by NT test………………	13
2.15 Lymphocyte proliferative responses to PRRSV……………………….	14
3. Results:	
3.1 Cloning and sequencing of cDNA of the ORFs 4, 5, 6 and 7 of PRRSV strain
    KB3.        ……………………………………………………………...	15
3.2 Transient expression of the cloned ORF4, 5, 6 and 7 genes by IHC
    staining………………….…………………………………………..…..	15
3.3 Construction of the pDelta-gD/KB3-orf4, 5, 6 and 7 vectors…………..	16
3.4 Detection of recombinant PrV-CT4, 5, 6 and 7 by PCR………………..	16
3.5 Transgene expression in Vero cells infected with recombinant PrV……	16
3.6 Detection of antibody responses by ELISA…...………………………..	17
3.7 Detection of anti-PRRSV antibodies by NT test……………………….	17
3.8 Induction of lymphocyte proliferative responses in mice by immunization with recombinant PrV…………………………………...	18
4. Discussion……………………………………………………………………..	19
References………………………………………………………………………..	22
Figures……………………………………………………………………………	25
Table……………………………………………………………………………...	43
Appendix…………………………………………………………………………	46
自述
                                    

1. Bastos, R. G., O. A. Dellagostin, R. G. Barletta, A. R. Doster, E. Nelson, and F. A. Osorio. 2002. Construction and immunogenicity of recombinant Mycobacterium bovis BCG expressing GP5 and M protein of porcine reproductive respiratory syndrome virus. Vaccine 21:21-29.
2. Bautista, E. M., P. Suarez, and T. W. Molitor. 1999. T cell responses to the structural polypeptides of porcine reproductive and respiratory syndrome virus. Arch. Virol. 144:117-134.
3. Christopher-Hennings, J., E. A. Nelson, J. K. Nelson, and D. A. Benfield. 1997. Effects of a modified-live virus vaccine against porcine reproductive and respiratory syndrome in boars. Am. J. Vet. Res. 58:40-45.
4. Cordero, O. J., C. S. Sarandeses, J. L. Lopez, E. Cancio, B. J. Regueiro, and M. Nogueira. 1991. Prothymosin alpha enhances interleukin 2 receptor expression in normal human T-lymphocytes. Int. J. Immunopharmacol. 13:1059-1065.
5. Cordero, O. J., C. S. Sarandeses, J. L. Lopez, and M. Nogueira. 1992. Prothymosin alpha enhances human natural killer cell cytotoxicity: role in mediating signals for NK activity. Lymphokine Cytokine Res. 11:277-285.
6. Haritos, A. A., G. J. Goodall, and B. L. Horecker. 1984. Prothymosin alpha: isolation and properties of the major immunoreactive form of thymosin alpha 1 in rat thymus. Proc. Natl. Acad. Sci. U. S. A 81:1008-1011.
7. Heffner, S., F. Kovacs, B. G. Klupp, and T. C. Mettenleiter. 1993. Glycoprotein gp50-negative pseudorabies virus: a novel approach toward a nonspreading live herpesvirus vaccine. J. Virol. 67:1529-1537.
8. Kimman, T. G., N. de Wind, T. de Bruin, Y. de Visser, and J. Voermans. 1994. Inactivation of glycoprotein gE and thymidine kinase or the US3-encoded protein kinase synergistically decreases in vivo replication of pseudorabies virus and the induction of protective immunity. Virology 205:511-518.
9. Kwang, J., F. Zuckermann, G. Ross, S. Yang, F. Osorio, W. Liu, and S. Low. 1999. Antibody and cellular immune responses of swine following immunisation with plasmid DNA encoding the PRRS virus ORF's 4, 5, 6 and 7. Res. Vet. Sci. 67:199-201.
10. McGregor, S., B. C. Easterday, A. S. Kaplan, and T. Ben Porat. 1985. Vaccination of swine with thymidine kinase-deficient mutants of pseudorabies virus. Am. J. Vet. Res. 46:1494-1497.
11. Meng, X. J. 2000. Heterogeneity of porcine reproductive and respiratory syndrome virus: implications for current vaccine efficacy and future vaccine development. Vet. Microbiol. 74:309-329.
12. Meulenberg, J. J., A. P. van Nieuwstadt, A. Essen-Zandbergen, and J. P. Langeveld. 1997. Posttranslational processing and identification of a neutralization domain of the GP4 protein encoded by ORF4 of Lelystad virus. J. Virol. 71:6061-6067.
13. Mulder, W., J. Pol, T. Kimman, G. Kok, J. Priem, and B. Peeters. 1996. Glycoprotein D-negative pseudorabies virus can spread transneuronally via direct neuron-to-neuron transmission in its natural host, the pig, but not after additional inactivation of gE or gI. J. Virol. 70:2191-2200.
14. Mulder, W. A., L. Jacobs, J. Priem, G. L. Kok, F. Wagenaar, T. G. Kimman, and J. M. Pol. 1994. Glycoprotein gE-negative pseudorabies virus has a reduced capability to infect se. J. Gen. Virol. 75 ( Pt 11):3095-3106.
15. Mulder, W. A., J. Priem, J. M. Pol, and T. G. Kimman. 1995. Role of viral proteins and concanavalin A in in vitro replication of pseudorabies virus in porcine peripheral blood mononuclear cells. J. Gen. Virol. 76 ( Pt 6):1433-1442.
16. Ostrowski, M., J. A. Galeota, A. M. Jar, K. B. Platt, F. A. Osorio, and O. J. Lopez. 2002. Identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus GP5 ectodomain. J. Virol. 76:4241-4250.
17. Peeters, B., A. Bouma, T. de Bruin, R. Moormann, A. Gielkens, and T. Kimman. 1994. Non-transmissible pseudorabies virus gp50 mutants: a new generation of safe live vaccines. Vaccine 12:375-380.
18. Peeters, B., N. de Wind, M. Hooisma, F. Wagenaar, A. Gielkens, and R. Moormann. 1992. Pseudorabies virus envelope glycoproteins gp50 and gII are essential for virus penetration, but only gII is involved in membrane fusion. J. Virol. 66:894-905.
19. Peeters, B., J. Pol, A. Gielkens, and R. Moormann. 1993. Envelope glycoprotein gp50 of pseudorabies virus is essential for virus entry but is not required for viral spread in mice. J. Virol. 67:170-177.
20. Pirzadeh, B. and S. Dea. 1997. Monoclonal antibodies to the ORF5 product of porcine reproductive and respiratory syndrome virus define linear neutralizing determinants. J. Gen. Virol. 78 ( Pt 8):1867-1873.
21. Pirzadeh, B. and S. Dea. 1998. Immune response in pigs vaccinated with plasmid DNA encoding ORF5 of porcine reproductive and respiratory syndrome virus. J. Gen. Virol. 79 ( Pt 5):989-999.
22. Rodriguez, M. J., J. Sarraseca, J. Fominaya, E. Cortes, A. Sanz, and J. I. Casal. 2001. Identification of an immunodominant epitope in the C terminus of glycoprotein 5 of porcine reproductive and respiratory syndrome virus. J. Gen. Virol. 82:995-999.
23. Rossow, K. D., D. A. Benfield, S. M. Goyal, E. A. Nelson, J. Christopher-Hennings, and J. E. Collins. 1996. Chronological immunohistochemical detection and localization of porcine reproductive and respiratory syndrome virus in gnotobiotic pigs. Vet. Pathol. 33:551-556.
24. Shiau, A. L., C. W. Liu, S. Y. Wang, C. Y. Tsai, and C. L. Wu. 2002. A simple selection system for construction of recombinant gD-negative pseudorabies virus as a vaccine vector. Vaccine 20:1186-1195.
25. Weiland, E., M. Wieczorek-Krohmer, D. Kohl, K. K. Conzelmann, and F. Weiland. 1999. Monoclonal antibodies to the GP5 of porcine reproductive and respiratory syndrome virus are more effective in virus neutralization than monoclonal antibodies to the GP4. Vet. Microbiol. 66:171-186.
26. Wootton, S., G. Koljesar, L. Yang, K. J. Yoon, and D. Yoo. 2001. Antigenic importance of the carboxy-terminal beta-strand of the porcine reproductive and respiratory syndrome virus nucleocapsid protein. Clin. Diagn. Lab Immunol. 8:598-603.
27. Yang, L., M. L. Frey, K. J. Yoon, J. J. Zimmerman, and K. B. Platt. 2000. Categorization of North American porcine reproductive and respiratory syndrome viruses: epitopic profiles of the N, M, GP5 and GP3 proteins and susceptibility to neutralization. Arch. Virol. 145:1599-1619.

校內：2006-09-09公開
校外：2006-09-09公開

簡易檢索 / 詳目顯示

相關論文