臺灣博碩士論文加值系統

牛病毒性下痢病毒（Bovine viral diarrhea virus, BVDV）是造成全世界牛群疾病的主要病原體，並對其對生產和一般健康狀況的不利影響造成重大經濟損失。 接種疫苗廣泛用於控制全世界BVDV感染的表現，但是BVDV野毒株的異質性和BVDV建立持續感染的獨特能力限制了疫苗的有效性。
在這項研究中，我們為台灣BVDV野生型構建了一種新的重組pET24a-BVDV-E2，其表達BVDV具有高免疫原性E2蛋白。使用大腸桿菌表達系統(Escherichia coli expression system)來表達這種重組蛋白並 Western blotting驗證E2蛋白的抗原性，然後將E2蛋白與ISA 206佐劑混合製成疫苗。我們已經確認注射小鼠疫苗的功效，雖然E2亞單位疫苗沒有達到預期的效果，但E2蛋白與少量滅活疫苗混合可提高滅活疫苗的有效性。 因此，我們推測rE2蛋白可能可以增強滅活BVDV疫苗效力。

Bovine viral diarrhea virus (BVDV) is a major pathogen responsible for diseases in cattle population worldwide and causes substantial economic losses for its negative effect on production and general health conditions. Vaccination is widely used to control manifestations of BVDV infections in the world, but in the heterogeneity of BVDV field strains and the unique ability of BVDV to establish persistent infections, limiting the effectiveness of the vaccine. In this study, we have constructed a novel recombinant pET24a-BVDV- E2 for Taiwan BVDV wild-type expressing the most immunogenic E2 antigen of BVDV. This recombinant protein was expressed by Escherichia coli expression system. The expression of E2 protein was verified by Western blotting. And then the E2 protein was mixed with ISA 206 adjuvent to produce subunit vaccine. We have confirmed the efficacy of the vaccine by Injected mice, the E2 subunit vaccine does not deliver on expectations, But the E2 protein mixed with a small amount of inactivated vaccine improve the effectiveness of inactivated vaccines. Therefore, we speculate that rE2 protein perhaps be an effective way to enhance the protective efficacy of inactivated BVDV vaccines.

目錄
第1章 緒言 1
第2章 文獻探討 3
2.1牛病毒性下痢病毒背景 3
2.2牛病毒性下痢病毒結構和分型 4
2.3傳播病毒 5
2.4臨床症狀 6
2.4.1急性感染 6
2.4.2胎兒感染 7
2.4.3持續感染 7
2.4.4粘膜疾病 8
2.5預防控制方法 8
2.6使用疫苗接種控制BVDV的情況 8
2.7 BVDV疫苗的發展 9
2.8本研究的目標與方向 11
第3章 材料與方法 13
3.1基因片段來源 13
3.2 E2基因片段 13
3.2.1設計BVDV-E2引子基因序列(Table 1) 13
3.2.2 聚合酶連鎖反應(Polymerase chain reaction, PCR) 13
3.2.3 膠體電泳法確認 PCR 產物大小 13
3.2.4 BVDV-E2基因片段純化 14
3.3 建構大腸桿菌表現系統 14
3.3.1 限制酶酵素切割 (Enzyme digestion) 14
3.3.2 黏合pET24a vector和BVDV-E2 (Ligation) 14
3.3.3 pET24a- BVDV-E2轉型至勝任細胞 DH5α (Transformation) 15
3.3.5 抽取pET24a- BVDV-E2質體 15
3.4 表現 BVDV-E2蛋白 15
3.4.1 pET24a- BVDV-E2 轉型至勝任細胞 BL21 15
3.4.2 BL21- pET24a- BVDV-E2 小量誘導蛋白測試 16
3.4.3 BL21- pET24a- BVDV-E2 大量誘導蛋白 16
3.5 蛋白質分析、純化與定量 17
3.5.1十二烷基硫酸鈉聚丙烯醯胺膠體(SDS-PAGE)製備 17
3.5.2重組 BVDV-E2蛋白進行SDS-PAGE電泳分析 17
3.5.3 重組 BVDV-E2蛋白進行抗原分析-西方墨點方(Western blot) 18
3.5.4 純化重組 BVDV-E2蛋白 18
3.5.5 重組 BVDV-E2蛋白透析 19
3.5.6 重組 BVDV-E2 蛋白定量 19
3.6 疫苗製作 19
3.7 動物試驗 20
3.7.1 小鼠免疫 20
3.7.2 檢測小鼠血清 Total IgG 20
3.7.3 小鼠脾臟細胞製備 21
3.7.4 淋巴細胞之計數 21
3.7.5 萃取小鼠脾臟細胞 RNA 22
3.7.8即時聚合酶連鎖反應(Quantitative real time polymerase chain reaction, Q-PCR) 22
第4章 結果 24
4.1 pET24a- BVDV-E2質體建構 24
4.2大腸桿菌表現BVDV-E2條件測試及抗原分析 24
4.3 BVDV-E2蛋白 24
4.4 重組 BVDV-E2蛋白透析 25
4.5重組 BVDV-E2蛋白純化回收率 25
4.6 第一次動物實驗 25
4.7 第二次動物實驗 26
第5章 討論 45
第6章 參考文獻 49
作者簡介 56

中華民國《動物用藥品檢驗標準》106年版.
Yesilbag K, Alpay G, Becher P. Variability and Global Distribution of Subgenotypes of Bovine Viral Diarrhea Virus. Viruses. 2017;9.
Cibulski S, Rivera-Patron M, Suárez N, Pirez M, Rossi S, Yendo AC, et al. Leaf saponins of Quillaja brasiliensis enhance long-term specific immune responses and promote dose-sparing effect in BVDV experimental vaccines. Vaccine. 2018;36:55-65.
Ridpath JF. Immunology of BVDV vaccines. Biologicals. 2013;41:14-9.
Tautz N, Meyers G, Thiel H-J. Pathogenesis of mucosal disease, a deadly disease of cattle caused by a pestivirus. Clinical and Diagnostic Virology. 1998;10:121-7.
Tajima M, Frey H-R, Yamato O, Maede Y, Moennig V, Scholz H, et al. Prevalence of genotypes 1 and 2 of bovine viral diarrhea virus in Lower Saxony, Germany. Virus Research. 2001;76:31-42.
Ridpath JF, Bolin SR, Dubovi EJ. Segregation of Bovine Viral Diarrhea Virus into Genotypes. Virology. 1994;205:66-74.
Ridpath JF, Bolin SR. Delayed Onset Postvaccinal Mucosal Disease as a Result of Genetic Recombination between Genotype 1 and Genotype 2 BVDV. Virology. 1995;212:259-62.
Peterhans E, Zanoni R, Bertoni G. How to succeed as a virus: strategies for dealing with the immune system. Veterinary Immunology and Immunopathology. 1999;72:111-7.
Paton DJ, Lowings JP, Barrett ADT. Epitope mapping of the gp53 envelope protein of bovine viral diarrhea virus. Virology. 1992;190:763-72.
Paton DJ. Pestivirus diversity. Journal of Comparative Pathology. 1995;112:215-36.
Nelson G, Marconi P, Periolo O, La Torre J, Alvarez MA. Immunocompetent truncated E2 glycoprotein of bovine viral diarrhea virus (BVDV) expressed in Nicotiana tabacum plants: A candidate antigen for new generation of veterinary vaccines. Vaccine. 2012;30:4499-504.
Meyers G, Thiel H-J. Molecular Characterization of Pestiviruses. In: Maramorosch K, Murphy FA, Shatkin AJ, editors. Advances in Virus Research: Academic Press; 1996. p. 53-118.
Liebler-Tenorio EM, Ridpath JF, Neill JD. Lesions and tissue distribution of viral antigen in severe acute versus subclinical acute infection with BVDV2. Biologicals. 2003;31:119-22.
Hornberg A, Fernández SR, Vogl C, Vilcek S, Matt M, Fink M, et al. Genetic diversity of pestivirus isolates in cattle from Western Austria. Veterinary Microbiology. 2009;135:205-13.
Greiser-Wilke I, Grummer B, Moennig V. Bovine viral diarrhoea eradication and control programmes in Europe. Biologicals. 2003;31:113-8
González AM, Arnaiz I, Yus E, Eiras C, Sanjuán M, Diéguez FJ. Evaluation of long-term antibody responses to two inactivated bovine viral diarrhoea virus (BVDV) vaccines. The Veterinary Journal. 2014;199:424-8.
Fulton RW, Step DL, Ridpath JF, Saliki JT, Confer AW, Johnson BJ, et al. Response of calves persistently infected with noncytopathic bovine viral diarrhea virus (BVDV) subtype 1b after vaccination with heterologous BVDV strains in modified live virus vaccines and Mannheimia haemolytica bacterin-toxoid. Vaccine. 2003;21:2980-5.
Evermann JF, Ridpath JF. Clinical and epidemiologic observations of bovine viral diarrhea virus in the northwestern United States. Veterinary Microbiology. 2002;89:129-39.
Deregt D, Bolin SR, van den Hurk J, Ridpath JF, Gilbert SA. Mapping of a type 1-specific and a type-common epitope on the E2 (gp53) protein of bovine viral diarrhea virus with neutralization escape mutants. Virus Research. 1998;53:81-90.
Dekker A, Wensvoort G, Terpstra C. Six antigenic groups within the genus pestivirus as identified by cross neutralization assays. Veterinary Microbiology. 1995;47:317-29.
Collett MS, Larson R, Gold C, Strick D, Anderson DK, Purchio AF. Molecular cloning and nucleotide sequence of the pestivirus bovine viral diarrhea virus. Virology. 1988;165:191-9.
Collett MS, Larson R, Belzer SK, Retzel E. Proteins encoded by bovine viral diarrhea virus: The genomic organization of a pestivirus. Virology. 1988;165:200-8.
Bruschke CJM, van Rijn PA, Moormann RJM, van Oirschot JT. Antigenically different pestivirus strains induce congenital infection in sheep: a model for bovine virus diarrhea virus vaccine efficacy studies. Veterinary Microbiology. 1996;50:33-43.
Bruschke CJM, Moormann RJM, van Oirschot JT, van Rijn PA. A subunit vaccine based on glycoprotein E2 of bovine virus diarrhea virus induces fetal protection in sheep against homologous challenge. Vaccine. 1997;15:1940-5.
Bachofen C, Stalder H, Braun U, Hilbe M, Ehrensperger F, Peterhans E. Co-existence of genetically and antigenically diverse bovine viral diarrhoea viruses in an endemic situation. Veterinary Microbiology. 2008;131:93-102.
Arias P, Orlich M, Prieto M, Cedillo Rosales S, Thiel HJ, Álvarez M, et al. Genetic heterogeneity of bovine viral diarrhoea viruses from Spain. Veterinary Microbiology. 2003;96:327-36.
Beer M, Hehnen H-R, Wolfmeyer A, Poll G, Kaaden O-R, Wolf G. A new inactivated BVDV genotype I and II vaccine: An immunisation and challenge study with BVDV genotype I. Veterinary Microbiology. 2000;77:195-208.
Bolin SR. The Pathogenesis of Mucosal Disease. Veterinary Clinics of North America: Food Animal Practice. 1995;11:489-500.
Brock KV, Lapin DR, Skrade DR. Embryo transfer from donor cattle persistently infected with bovine viral diarrhea virus. Theriogenology. 1997;47:837-44.
Brock KV, Stringfellow D. Comparative effects of cytopathic and noncytopathic bovine viral diarrhea virus on bovine blastocysts. Theriogenology. 1993;39:196.
Fulton RW, Ridpath JF, Confer AW, Saliki JT, Burge LJ, Payton ME. Bovine viral diarrhoea virus antigenic diversity: impact on disease and vaccination programmes. Biologicals. 2003;31:89-95.
Givens MD, Marley MS. Immunology of chronic BVDV infections. Biologicals. 2013;41:26-30.
Grooms DL. Reproductive consequences of infection with bovine viral diarrhea virus. Veterinary Clinics of North America: Food Animal Practice. 2004;20:5-19.
Houe H. Survivorship of animals persistently infected with bovine virus diarrhoea virus (BVDV). Preventive Veterinary Medicine. 1993;15:275-83.
Houe H. Epidemiological features and economical importance of bovine virus diarrhoea virus (BVDV) infections. Veterinary Microbiology. 1999;64:89-107.
Houe H. Economic impact of BVDV infection in dairies. Biologicals. 2003;31:137-43.
Howard CJ, Brownlie J, Clarke MC. Comparison by the neutralisation assay of pairs of non-cytopathogenic and cytopathogenic strains of bovine virus diarrhoea virus isolated from cases of mucosal disease. Veterinary Microbiology. 1987;13:361-9.
Howard CJ, Clarke MC, Brownlie J. An Enzyme-Linked Immunosorbent Assay (ELISA) for the detection of antibodies to Bovine Viral Diarrhoea Virus (BVDV) in cattle sera. Veterinary Microbiology. 1985;10:359-69.
Howard CJ, Clarke MC, Brownlie J. Protection against respiratory infection with bovine virus diarrhoea virus by passively acquired antibody. Veterinary Microbiology. 1989;19:195-203.
Kelling CL. Evolution of bovine viral diarrhea virus vaccines. Veterinary Clinics of North America: Food Animal Practice. 2004;20:115-29.
Lindberg ALE, Alenius S. Principles for eradication of bovine viral diarrhoea virus (BVDV) infections in cattle populations. Veterinary Microbiology. 1999;64:197-222.
Meyling A, Mikél Jensen A. Transmission of bovine virus diarrhoea virus (BVDV) by artificial insemination (AI) with semen from a persistently-infected bull. Veterinary Microbiology. 1988;17:97-105.
Moennig V, Eicken K, Flebbe U, Frey HR, Grummer B, Haas L, et al. Implementation of two-step vaccination in the control of bovine viral diarrhoea (BVD). Preventive Veterinary Medicine. 2005;72:109-14.
Newcomer BW, Chamorro MF, Walz PH. Vaccination of cattle against bovine viral diarrhea virus. Veterinary Microbiology. 2017;206:78-83.
Newcomer BW, Givens D. Diagnosis and Control of Viral Diseases of Reproductive Importance: Infectious Bovine Rhinotracheitis and Bovine Viral Diarrhea. Veterinary Clinics of North America: Food Animal Practice. 2016;32:425-41.
Palomares RA, Marley SM, Givens MD, Gallardo RA, Brock KV. Bovine viral diarrhea virus fetal persistent infection after immunization with a contaminated modified-live virus vaccine. Theriogenology. 2013;79:1184-95.
Pande A, Carr BV, Wong SYC, Dalton K, Jones IM, McCauley JW, et al. The glycosylation pattern of baculovirus expressed envelope protein E2 affects its ability to prevent infection with bovine viral diarrhoea virus. Virus Research. 2005;114:54-62.
Raue R, Harmeyer SS, Nanjiani IA. Antibody responses to inactivated vaccines and natural infection in cattle using bovine viral diarrhoea virus ELISA kits: Assessment of potential to differentiate infected and vaccinated animals. The Veterinary Journal. 2011;187:330-4.
Ridpath J. The Contribution of Infections with Bovine Viral Diarrhea Viruses to Bovine Respiratory Disease. Veterinary Clinics of North America: Food Animal Practice. 2010;26:335-48.
Ridpath JF. Bovine Viral Diarrhea Virus A2 - Mahy, Brian W.J. In: Regenmortel MHVV, editor. Encyclopedia of Virology (Third Edition). Oxford: Academic Press; 2008. p. 374-80.
Thomas C, Young NJ, Heaney J, Collins ME, Brownlie J. Evaluation of efficacy of mammalian and baculovirus expressed E2 subunit vaccine candidates to bovine viral diarrhoea virus. Vaccine. 2009;27:2387-93.
Viet A-F, Fourichon C, Seegers H, Jacob C, Guihenneuc-Jouyaux C. A model of the spread of the bovine viral-diarrhoea virus within a dairy herd. Preventive Veterinary Medicine. 2004;63:211-36.
Wernike K, Schirrmeier H, Strebelow H-G, Beer M. Eradication of bovine viral diarrhea virus in Germany—Diversity of subtypes and detection of live-vaccine viruses. Veterinary Microbiology. 2017;208:25-9.
Windeyer MC, Leslie KE, Godden SM, Hodgins DC, Lissemore KD, LeBlanc SJ. The effects of viral vaccination of dairy heifer calves on the incidence of respiratory disease, mortality, and growth. Journal of Dairy Science. 2012;95:6731-9.
Donis RO. Molecular Biology of Bovine Viral Diarrhea Virus and its Interactions with the Host. Veterinary Clinics of North America: Food Animal Practice. 1995;11:393-423.
Marzocca MP, Seki C, Giambiagi SM, Robiolo B, Schauer R, Dus Santos MJ, et al. Truncated E2 of bovine viral diarrhea virus (BVDV) expressed in Drosophila melanogaster cells: A candidate antigen for a BVDV ELISA. Journal of Virological Methods. 2007;144:49-56.
Brodersen BW. Bovine viral diarrhea virus infections: manifestations of infection and recent advances in understanding pathogenesis and control. Vet Pathol. 2014;51:453-64.
Brackenbury LS, Carr BV, Charleston B. Aspects of the innate and adaptive immune responses to acute infections with BVDV. Veterinary Microbiology. 2003;96:337-44.
Alves PA, Figueiredo PO, de Oliveira CHS, Barbosa JD, Lima DHS, Bomjardim HA, et al. Occurrence of Pseudocowpox virus associated to Bovine viral diarrhea virus-1, Brazilian Amazon. Comp Immunol Microbiol Infect Dis. 2016;49:70-5.
Li T, Huang M, Xiao H, Zhang G, Ding J, Wu P, et al. Selection and characterization of specific nanobody against bovine virus diarrhea virus (BVDV) E2 protein. PLoS One. 2017;12:e0178469.
Rajput MKS, Abdelsalam K, Darweesh MF, Braun LJ, Kerkvliet J, Hoppe AD, et al. Both cytopathic and non-cytopathic bovine viral diarrhea virus (BVDV) induced autophagy at a similar rate. Veterinary Immunology and Immunopathology. 2017;193-194:1-9.
Bielefeldt-Ohmann H, Tolnay AE, Reisenhauer CE, Hansen TR, Smirnova N, Van Campen H. Transplacental infection with non-cytopathic bovine viral diarrhoea virus types 1b and 2: viral spread and molecular neuropathology. J Comp Pathol. 2008;138:72-85.
Bhuyan AA, Memon AM, Bhuiyan AA, Zhonghua L, Zhang B, Ye S, et al. The construction of recombinant Lactobacillus casei expressing BVDV E2 protein and its immune response in mice. Journal of Biotechnology. 2018;270:51-60.
da Silva Cardoso Pinto V, Alves MF, de Souza Nunes Martins M, Basso AC, Tannura JH, Pontes JHF, et al. Effects of oocytes exposure to bovine diarrhea viruses BVDV-1, BVDV-2 and Hobi-like virus on in vitro-produced bovine embryo development and viral infection. Theriogenology. 2017;97:67-72.
Chase CCL. The impact of BVDV infection on adaptive immunity. Biologicals. 2013;41:52-60.
Platt R, Coutu C, Meinert T, Roth JA. Humoral and T cell-mediated immune responses to bivalent killed bovine viral diarrhea virus vaccine in beef cattle. Veterinary Immunology and Immunopathology. 2008;122:8-15.
Lanyon SR, Hill FI, Reichel MP, Brownlie J. Bovine viral diarrhoea: Pathogenesis and diagnosis. The Veterinary Journal. 2014;199:201-9.
Downey-Slinker ED, Ridpath JF, Sawyer JE, Skow LC, Herring AD. Antibody titers to vaccination are not predictive of level of protection against a BVDV type 1b challenge in Bos indicus - Bos taurus steers. Vaccine. 2016;34:5053-9.