臺灣博碩士論文加值系統

豬流行性下痢 ( Porcine epidemic diarrhea, PED )，是由豬流行性下痢病毒 ( Porcine epidemic diarrhea virus, PEDV ) 感染所導致，於全年齡豬隻皆會感染，感染後會在豬隻身上引起下痢、脫水、嘔吐及食慾不振等症狀。PED病例首次於西元1971年於英格蘭被注意，病毒則在西元1978年於比利時被分離出來。此病毒屬於冠狀病毒科 ( family Coronaviridae )的甲型冠狀病毒屬 ( genus Alphacoronavirus )，屬於單股RNA病毒，基因體長度約為28kb，能夠轉錄出四個結構蛋白，分別為大小約180-260kDa的棘突蛋白 ( Spike protein, S )、27-32kDa的膜蛋白 ( Membrane protein, M )、7kDa的封套蛋白 ( Envelope protein, E )、及55-58kDa的核蛋白 ( Nucleocapsid protein,N )。而其中S蛋白主要與受器結合並帶領病毒進入細胞，而且能夠於宿主體內誘發中和性抗體，也證實S蛋白上具有中和性抗原決定位 ( neutralizating epitopes )。另一方面，短芽孢菌 ( Brevibacillus choshinensis )目前常用來進行蛋白質的表現，此表線系統為革蘭氏陽性菌表現系統，優點在於其質體上帶有訊號胜肽 ( signal peptide )，能直接將目標蛋白分泌到培養液中，使我們在生產蛋白質時，可以不用進行破菌而得到產物。另外，此系統具有雙硫鍵的修飾，能使蛋白質產生摺疊，而更佳符合目標蛋白質原先的天然構形及可溶性。我們將以此陽性菌表現系統來生產PEDV的S蛋白作為次單位疫苗，並透過十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 ( SDS-PAGE )和西方墨點法 ( Western Blot )確認蛋白是可以以此表現系統表現的。搭配水包油包水佐劑 ( Water-in-oil-in-water, W/O/W )免疫於小鼠，接著進行酵素免疫分析 ( ELISA )及中和試驗 ( NT )，以評估此次單位疫苗的免疫能力，期望能對於PEDV具有保護性。

Porcine epidemic diarrhea (PED) is caused by porcine epidemic diarrhea virus (PEDV). It would infect all age of swines, and be characterized by severe diarrhea, vomiting, and dehydration. PED was first reported in England in 1971 and the virus was first identified in Belgium. The virus belongs to the genus Alphacoronavirus, family Coronaviridae. It`s genome is single-stranded, positive-sense RNA which is contain 28 kb in length. The genome can translate four structure proteins, i.e. spike protein (S, 180-220kDa), membrane protein (M, 27-32kDa), envelope protein (E, 7kDa), and nucleocapsid protein (N, 55-58kDa). The spike protein plays the important role in receptor binding activity and elicts neutralizating antibody. And there are some studies prove evidences that spike protein contain neutralizating epitopes. In addition, Brevibacillus choshinensis expression system was use to produce recombinant protein in recent year. It is the gram-postive bacteria. Because the expression vector contains signal peptide, it can produce the recombinant protein in secreted form, and then we can harvest the protein without lysis bacteria and minimize lipopolysaccharide (LPS). B. choshinensis expression system has the ability to form a disulfide bond, which led the protein`s conformation close to native form. In this study, we used B. choshinensis expression system to produce PEDV spike protein to develop subunit vaccine, and through the result of SDS and western blot the protein could be produced by gram-positive bacterial expression system . We immunize the mice and harvest the serum to dectet the immune response by ELISA and neutralizating test.

目錄
目錄
中文摘要 I
Abstract I
謝誌 III
目錄 I
圖表目錄 I
第一章 緒言 3
第2章 文獻回顧 5
2.1 豬流行性下痢病毒（Porcine epidemic diarrhea ,PED） 5
2.1.1 PED流行病學 5
2.1.2 PEDV之傳播 6
2.1.3臨床症狀 6
2.1.4免疫機轉 7
2.1.5疾病檢測 7
2.1.5.1酵素免疫吸附分析法（enzyme-linked immunosorbent assay, ELISA） 7
2.1.5.2電子顯微鏡（electron microscopy） 8
2.1.5.3反轉錄聚合酶鍊鎖反應（reverse transcription-polymerase chain reaction, RT-PCR） 8
2.1.5.4細胞培養 8
2.1.5.5免疫組織化學法（immunohistochemical techniques） 8
2.1.6 預防與控制 8
2.1.6.1反飼方式 9
2.1.6.2接種疫苗 9
2.1.6.3環境消毒 9
2.1.7病毒形態、結構與生長特性 10
2.1.7.1病毒培養 11
2.1.7.2血球凝集現象 11
2.1.8宿主 11
2.2 次單位疫苗 11
2.2.1 大腸桿菌表現系統（E.coli expression system） 12
2.2.1.1　IPTG誘導 13
2.2.2　短芽孢桿菌格蘭氏陽性表現系統（ Brevibacillus choshinensis expression system） 13
2.2.2.1　背景介紹 13
2.2.2.2　短芽孢桿菌重組蛋白表現系統（ Brevibacillus choshinensis expression system） 14
第3章 材料方法 15
3.1 實驗方法與架構 15
3.2病材及組織來源 15
3.4病毒培養 15
3.4.1 細胞培養 15
3.4.2細胞的冷凍保存 16
3.4.3 病毒擴增 16
3.4.4 抽取PEDV的RNA 16
3.4.5 病毒RNA的cDNA製備 – 反轉錄作用 17
3.5.2 Q-PCR 17
3.4.6 建立病毒定量標準曲線 18
3.5 抗原製備 19
3.5.1 PEDV spike 基因轉殖（cloning）用引子 19
3.5.2 DNA 電泳分析 19
3.5.3 PCR 產物膠體回收 20
3.5.5 接合作用( ligation ) 20
3.5.4 E.coli的轉型作用（Transformation） 20
3.5.4.1 塗盤及挑菌 21
3.5.4.2 細菌的保存與小量質體 DNA 萃取 21
3.5.5 將目標基因轉殖到表現載體pET-24a並轉型至E.coli 22
3.5.6將目標基因轉殖到pNC-His表現載體 22
3.5.7將目標基因轉型至B. choshinensis 22
3.5.5 E.coli expression system蛋白質的誘導 23
3.5.5.1 蛋白質的萃取 23
3.5.5.2 蛋白質的純化 23
3.5.6 B. choshinensis expression system 的蛋白質生產 24
3.5.7蛋白質的濃縮 24
3.5.7 蛋白質電泳(SDS- PAGE) 24
3.5.8 西方墨點法(Western blot) 25
3.6動物實驗 26
3.6.1試驗小鼠 26
3.6.2免疫試驗及動物實驗組別 26
3.6.3 酵素免疫分析法（Enzyme-linked immunosorbent assay；ELISA） 26
3.6.4 血清中和試驗 27
第4章　結果 28
4.1 E.coli expression system基因轉殖、轉形檢測及蛋白質表現 28
4.1.1 PED spike protein cDNA製備 28
4.1.2基因進行限制酶酵素處理及轉殖入pET-24a載體中 28
4.1.3 PCR確認pET24a-PED S protein是否成功轉型到BL21 28
4.1.4確認E.coli expression system重組蛋白的表現 29
4.1.4.1製備SDS gel並以西方墨點法確認pET24a-PED S protein可在大腸桿菌中表現 29
4.1.4.2以鎳離子親和性樹脂進行蛋白質的純化 29
4.2 B. choshinensis expression system 基因轉殖、轉形檢測及蛋白質表現 29
4.2.1目標基因進行限制酶酵素處理及轉殖入pNC-His載體中 29
4.2.2 PCR確認pNC-His - PED S protein是否成功轉型到B. choshinensis 30
4.2.3試表現重組蛋白質以確定挑的菌落是否會製造蛋白質 30
4.2.4確認B. choshinensis重組蛋白質的表現 30
4.2.4.1製備SDS gel並以西方墨點法確認pNC-His-PED S protein可在B. choshinensis中表現 30
4.2.4.2 B. choshinensis重組蛋白濃度測定 31
4.3將重組蛋白於小鼠進行動物實驗 31
4.3.1重組蛋白能引發體液性免疫反應中IgG之抗體 31
4.3.2重組蛋白能引起病毒中和性反應 31
4.4病毒定量標準曲線 31
第5章　討論 46
第6章　參考文獻 50

第6章　參考文獻

1. Appleyard G, and Maber HB. Plaque formation by influenza viruses in the presence of trypsin. The Journal of general virology 25: 351-357, 1974.
2. Callebaut P, Debouck P, and Pensaert M. Enzyme-linked immunosorbent assay for the detection of the coronavirus-like agent and its antibodies in pigs with porcine epidemic diarrhea. Veterinary microbiology 7: 295-306, 1982.
3. Carvajal A, Lanza I, Diego R, Rubio P, and Carmenes P. Evaluation of a blocking ELISA using monoclonal antibodies for the detection of porcine epidemic diarrhea virus and its antibodies. Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 7: 60-64, 1995.
4. Chasey D, and Cartwright SF. Virus-like particles associated with porcine epidemic diarrhoea. Research in veterinary science 25: 255-256, 1978.
5. Chen F, Pan Y, Zhang X, Tian X, Wang D, Zhou Q, Song Y, and Bi Y. Complete genome sequence of a variant porcine epidemic diarrhea virus strain isolated in China. Journal of virology 86: 12448, 2012a.
6. Chen J, Liu X, Shi D, Shi H, Zhang X, and Feng L. Complete genome sequence of a porcine epidemic diarrhea virus variant. Journal of virology 86: 3408, 2012b.
7. Chen J, Liu X, Shi D, Shi H, Zhang X, Li C, Chi Y, and Feng L. Detection and molecular diversity of spike gene of porcine epidemic diarrhea virus in China. Viruses 5: 2601-2613, 2013.
8. Chen J, Wang C, Shi H, Qiu H, Liu S, Chen X, Zhang Z, and Feng L. Molecular epidemiology of porcine epidemic diarrhea virus in China. Archives of virology 155: 1471-1476, 2010.
9. Chen Q, Li G, Stasko J, Thomas JT, Stensland WR, Pillatzki AE, Gauger PC, Schwartz KJ, Madson D, Yoon KJ, Stevenson GW, Burrough ER, Harmon KM, Main RG, and Zhang J. Isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the United States. Journal of clinical microbiology 52: 234-243, 2014a.
10. Chen X, Yang J, Yu F, Ge J, Lin T, and Song T. Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (PEDV) samples from field cases in Fujian, China. Virus genes 45: 499-507, 2012c.
11. Chen Y, Shi Y, Deng H, Gu T, Xu J, Ou J, Jiang Z, Jiao Y, Zou T, and Wang C. Characterization of the porcine epidemic diarrhea virus codon usage bias. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 28: 95-100, 2014b.
12. D'Urzo N, Martinelli M, Nenci C, Brettoni C, Telford JL, and Maione D. High-level intracellular expression of heterologous proteins in Brevibacillus choshinensis SP3 under the control of a xylose inducible promoter. Microbial cell factories 12: 12, 2013.
13. Dea S, Elazhary MA, Martineau GP, and Vaillancourt J. Parvovirus-like particles associated with diarrhea in unweaned piglets. Canadian journal of comparative medicine Revue canadienne de medecine comparee 49: 343-345, 1985.
14. Duarte M, and Laude H. Sequence of the spike protein of the porcine epidemic diarrhoea virus. The Journal of general virology 75 ( Pt 5): 1195-1200, 1994a.
15. Duarte M, Tobler K, Bridgen A, Rasschaert D, Ackermann M, and Laude H. Sequence analysis of the porcine epidemic diarrhea virus genome between the nucleocapsid and spike protein genes reveals a polymorphic ORF. Virology 198: 466-476, 1994b.
16. Enjuanes L, Smerdou C, Sanchez CM, Sune C, Kelly S, Curtiss R, 3rd, and Torres JM. Induction of transmissible gastroenteritis coronavirus specific immune responses using vectors with enteric tropism. Advances in experimental medicine and biology 371B: 1535-1541, 1995.
17. Fan JH, Zuo YZ, Li JH, and Pei LH. Heterogeneity in membrane protein genes of porcine epidemic diarrhea viruses isolated in China. Virus genes 45: 113-117, 2012.
18. Gao Y, Kou Q, Ge X, Zhou L, Guo X, and Yang H. Phylogenetic analysis of porcine epidemic diarrhea virus field strains prevailing recently in China. Archives of virology 158: 711-715, 2013.
19. Hofmann M, and Wyler R. Propagation of the virus of porcine epidemic diarrhea in cell culture. Journal of clinical microbiology 26: 2235-2239, 1988.
20. Horne I, Williams M, Sutherland TD, Russell RJ, and Oakeshott JG. A Brevibacillus choshinensis system that secretes cytoplasmic proteins. Journal of molecular microbiology and biotechnology 8: 81-90, 2004.
21. Horvath I, and Mocsari E. Ultrastructural changes in the small intestinal epithelium of suckling pigs affected with a transmissible gastroenteritis (TGE)-like disease. Archives of virology 68: 103-113, 1981.
22. Huang YW, Dickerman AW, Pineyro P, Li L, Fang L, Kiehne R, Opriessnig T, and Meng XJ. Origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the United States. mBio 4: e00737-00713, 2013.
23. Ishikawa K, Sekiguchi H, Ogino T, and Suzuki S. Direct and rapid detection of porcine epidemic diarrhea virus by RT-PCR. Journal of virological methods 69: 191-195, 1997.
24. Jung K, Kang BK, Kim JY, Shin KS, Lee CS, and Song DS. Effects of epidermal growth factor on atrophic enteritis in piglets induced by experimental porcine epidemic diarrhoea virus. Veterinary journal 177: 231-235, 2008.
25. Kim O, and Chae C. In situ hybridization for the detection and localization of porcine epidemic diarrhea virus in the intestinal tissues from naturally infected piglets. Veterinary pathology 37: 62-67, 2000.
26. Kocherhans R, Bridgen A, Ackermann M, and Tobler K. Completion of the porcine epidemic diarrhoea coronavirus (PEDV) genome sequence. Virus genes 23: 137-144, 2001.
27. Kweon CH, Kwon BJ, Lee JG, Kwon GO, and Kang YB. Derivation of attenuated porcine epidemic diarrhea virus (PEDV) as vaccine candidate. Vaccine 17: 2546-2553, 1999.
28. Kweon CH, Kwon BJ, Woo SR, Kim JM, Woo GH, Son DH, Hur W, and Lee YS. Immunoprophylactic effect of chicken egg yolk immunoglobulin (Ig Y) against porcine epidemic diarrhea virus (PEDV) in piglets. The Journal of veterinary medical science / the Japanese Society of Veterinary Science 62: 961-964, 2000.
29. Laude H, Van Reeth K, and Pensaert M. Porcine respiratory coronavirus: molecular features and virus-host interactions. Veterinary research 24: 125-150, 1993.
30. Lee S, and Lee C. Outbreak-related porcine epidemic diarrhea virus strains similar to US strains, South Korea, 2013. Emerging infectious diseases 20: 1223-1226, 2014.
31. Li Z, Chen F, Yuan Y, Zeng X, Wei Z, Zhu L, Sun B, Xie Q, Cao Y, Xue C, Ma J, and Bee Y. Sequence and phylogenetic analysis of nucleocapsid genes of porcine epidemic diarrhea virus (PEDV) strains in China. Archives of virology 158: 1267-1273, 2013.
32. Li ZL, Zhu L, Ma JY, Zhou QF, Song YH, Sun BL, Chen RA, Xie QM, and Bee YZ. Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (PEDV) field strains in south China. Virus genes 45: 181-185, 2012.
33. Lin CN, Chung WB, Chang SW, Wen CC, Liu H, Chien CH, and Chiou MT. US-like strain of porcine epidemic diarrhea virus outbreaks in Taiwan, 2013-2014. The Journal of veterinary medical science / the Japanese Society of Veterinary Science 76: 1297-1299, 2014.
34. Maehashi K, Matano M, Saito M, and Udaka S. Extracellular production of riboflavin-binding protein, a potential bitter inhibitor, by Brevibacillus choshinensis. Protein expression and purification 71: 85-90, 2010.
35. Miyauchi A, Ozawa M, Mizukami M, Yashiro K, Ebisu S, Tojo T, Fujii T, and Takagi H. Structural conversion from non-native to native form of recombinant human epidermal growth factor by Brevibacillus choshinensis. Bioscience, biotechnology, and biochemistry 63: 1965-1969, 1999.
36. Mizukami M, Hanagata H, and Miyauchi A. Brevibacillus expression system: host-vector system for efficient production of secretory proteins. Current pharmaceutical biotechnology 11: 251-258, 2010.
37. Mizukami M, Tokunaga H, Onishi H, Ueno Y, Hanagata H, Miyazaki N, Kiyose N, Ito Y, Ishibashi M, Hagihara Y, Arakawa T, Miyauchi A, and Tokunaga M. Highly efficient production of VHH antibody fragments in Brevibacillus choshinensis expression system. Protein expression and purification 105C: 23-32, 2014.
38. Mu T, Liang W, Ju Y, Wang Z, Wang Z, Roycik MD, Sang QX, Yu D, Xiang H, and Fang X. Efficient soluble expression of secreted matrix metalloproteinase 26 in Brevibacillus choshinensis. Protein expression and purification 91: 125-133, 2013.
39. Oh JS, Song DS, Yang JS, Song JY, Moon HJ, Kim TY, and Park BK. Comparison of an enzyme-linked immunosorbent assay with serum neutralization test for serodiagnosis of porcine epidemic diarrhea virus infection. Journal of veterinary science 6: 349-352, 2005.
40. Olanratmanee EO, Kunavongkrit A, and Tummaruk P. Impact of porcine epidemic diarrhea virus infection at different periods of pregnancy on subsequent reproductive performance in gilts and sows. Animal reproduction science 122: 42-51, 2010.
41. Panda AK, Bisht SS, DeMondal S, Senthil Kumar N, Gurusubramanian G, and Panigrahi AK. Brevibacillus as a biological tool: a short review. Antonie van Leeuwenhoek 105: 623-639, 2014.
42. Park JE, Cruz DJ, and Shin HJ. Trypsin-induced hemagglutination activity of porcine epidemic diarrhea virus. Archives of virology 155: 595-599, 2010.
43. Park SJ, Moon HJ, Luo Y, Kim HK, Kim EM, Yang JS, Song DS, Kang BK, Lee CS, and Park BK. Cloning and further sequence analysis of the ORF3 gene of wild- and attenuated-type porcine epidemic diarrhea viruses. Virus genes 36: 95-104, 2008.
44. Pensaert MB, and de Bouck P. A new coronavirus-like particle associated with diarrhea in swine. Archives of virology 58: 243-247, 1978.
45. Pijpers A, van Nieuwstadt AP, Terpstra C, and Verheijden JH. Porcine epidemic diarrhoea virus as a cause of persistent diarrhoea in a herd of breeding and finishing pigs. The Veterinary record 132: 129-131, 1993.
46. Puranaveja S, Poolperm P, Lertwatcharasarakul P, Kesdaengsakonwut S, Boonsoongnern A, Urairong K, Kitikoon P, Choojai P, Kedkovid R, Teankum K, and Thanawongnuwech R. Chinese-like strain of porcine epidemic diarrhea virus, Thailand. Emerging infectious diseases 15: 1112-1115, 2009.
47. Pyo HM, Kim IJ, Kim SH, Kim HS, Cho SD, Cho IS, and Hyun BH. Escherichia coli expressing single-chain Fv on the cell surface as a potential prophylactic of porcine epidemic diarrhea virus. Vaccine 27: 2030-2036, 2009.
48. Shibata I, Ono M, and Mori M. Passive protection against porcine epidemic diarrhea (PED) virus in piglets by colostrum from immunized cows. The Journal of veterinary medical science / the Japanese Society of Veterinary Science 63: 655-658, 2001.
49. Shibata I, Tsuda T, Mori M, Ono M, Sueyoshi M, and Uruno K. Isolation of porcine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of different ages. Veterinary microbiology 72: 173-182, 2000.
50. Shida O, Takagi H, Kadowaki K, and Komagata K. Proposal for two new genera, Brevibacillus gen. nov. and Aneurinibacillus gen. nov. International journal of systematic bacteriology 46: 939-946, 1996.
51. Song D, and Park B. Porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines. Virus genes 44: 167-175, 2012.
52. Song DS, Kang BK, Lee SS, Yang JS, Moon HJ, Oh JS, Ha GW, Jang YS, and Park BK. Use of an internal control in a quantitative RT-PCR assay for quantitation of porcine epidemic diarrhea virus shedding in pigs. Journal of virological methods 133: 27-33, 2006.
53. Song DS, Oh JS, Kang BK, Yang JS, Moon HJ, Yoo HS, Jang YS, and Park BK. Oral efficacy of Vero cell attenuated porcine epidemic diarrhea virus DR13 strain. Research in veterinary science 82: 134-140, 2007.
54. Song DS, Yang JS, Oh JS, Han JH, and Park BK. Differentiation of a Vero cell adapted porcine epidemic diarrhea virus from Korean field strains by restriction fragment length polymorphism analysis of ORF 3. Vaccine 21: 1833-1842, 2003.
55. Stevenson GW, Hoang H, Schwartz KJ, Burrough ER, Sun D, Madson D, Cooper VL, Pillatzki A, Gauger P, Schmitt BJ, Koster LG, Killian ML, and Yoon KJ. Emergence of Porcine epidemic diarrhea virus in the United States: clinical signs, lesions, and viral genomic sequences. Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 25: 649-654, 2013.
56. Sueyoshi M, Tsuda T, Yamazaki K, Yoshida K, Nakazawa M, Sato K, Minami T, Iwashita K, Watanabe M, Suzuki Y, and et al. An immunohistochemical investigation of porcine epidemic diarrhoea. Journal of comparative pathology 113: 59-67, 1995.
57. Sun R, Leng Z, Zhai SL, Chen D, and Song C. Genetic variability and phylogeny of current Chinese porcine epidemic diarrhea virus strains based on spike, ORF3, and membrane genes. TheScientificWorldJournal 2014: 208439, 2014.
58. Sun RQ, Cai RJ, Chen YQ, Liang PS, Chen DK, and Song CX. Outbreak of porcine epidemic diarrhea in suckling piglets, China. Emerging infectious diseases 18: 161-163, 2012.
59. Takahashi K, Okada K, and Ohshima K. An outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in Japan. Nihon juigaku zasshi The Japanese journal of veterinary science 45: 829-832, 1983.
60. Takano K, and Miller JR. Letter to the editor. Science 175: 474, 1972.
61. Tanaka R, Kosugi K, Mizukami M, Ishibashi M, Tokunaga H, and Tokunaga M. Expression and purification of thioredoxin (TrxA) and thioredoxin reductase (TrxB) from Brevibacillus choshinensis. Protein expression and purification 37: 385-391, 2004.
62. Tanaka R, Mizukami M, Ishibashi M, Tokunaga H, and Tokunaga M. Cloning and expression of the ccdA-associated thiol-disulfide oxidoreductase (catA) gene from Brevibacillus choshinensis: stimulation of human epidermal growth factor production. Journal of biotechnology 103: 1-10, 2003.
63. Tanio M, Kusunoki H, and Kohno T. (1)H, (1)(3)C and (1)(5)N backbone resonance assignments of the monomeric human M-ficolin fibrinogen-like domain secreted by Brevibacillus choshinensis. Biomolecular NMR assignments 8: 207-211, 2014.
64. Tanio M, Tanaka R, Tanaka T, and Kohno T. Amino acid-selective isotope labeling of proteins for nuclear magnetic resonance study: proteins secreted by Brevibacillus choshinensis. Analytical biochemistry 386: 156-160, 2009.
65. Tokunaga M, Mizukami M, Yamasaki K, Tokunaga H, Onishi H, Hanagata H, Ishibashi M, Miyauchi A, Tsumoto K, and Arakawa T. Secretory production of single-chain antibody (scFv) in Brevibacillus choshinensis using novel fusion partner. Applied microbiology and biotechnology 97: 8569-8580, 2013.
66. Turgeon DC, Morin M, Jolette J, Higgins R, Marsolais G, and DiFranco E. Coronavirus-like particles associated with diarrhea in baby pigs in Quebec. The Canadian veterinary journal La revue veterinaire canadienne 21: 100-xxiii, 1980.
67. van Nieuwstadt AP, and Zetstra T. Use of two enzyme-linked immunosorbent assays to monitor antibody responses in swine with experimentally induced infection with porcine epidemic diarrhea virus. American journal of veterinary research 52: 1044-1050, 1991.
68. Wang K, Lu W, Chen J, Xie S, Shi H, Hsu H, Yu W, Xu K, Bian C, Fischer WB, Schwarz W, Feng L, and Sun B. PEDV ORF3 encodes an ion channel protein and regulates virus production. FEBS letters 586: 384-391, 2012.
69. Wang L, Byrum B, and Zhang Y. New variant of porcine epidemic diarrhea virus, United States, 2014. Emerging infectious diseases 20: 917-919, 2014.
70. Wang QH, Costantini V, and Saif LJ. Porcine enteric caliciviruses: genetic and antigenic relatedness to human caliciviruses, diagnosis and epidemiology. Vaccine 25: 5453-5466, 2007.
71. Wood EN. Transmissible gastroenteritis and epidemic diarrhoea of pigs. The British veterinary journal 135: 305-314, 1979.
72. Woods RD. Efficacy of a transmissible gastroenteritis coronavirus with an altered ORF-3 gene. Canadian journal of veterinary research = Revue canadienne de recherche veterinaire 65: 28-32, 2001.
73. Yashiro K, Lowenthal JW, O'Neil TE, Ebisu S, Takagi H, and Moore RJ. High-level production of recombinant chicken interferon-gamma by Brevibacillus choshinensis. Protein expression and purification 23: 113-120, 2001.
74. 吳懷軒. 2012至2014年臺灣地區豬流行性下痢病毒之檢測與序列分析. 2014
75. 鄧明中. 淺談豬流行性下痢之現況與防治方法. 2014
76. 張惟淳. 雞球蟲次單位疫苗開發. 2015
77. 沈忻穎. 雞貧血病毒次單位疫苗及不活化疫苗免疫評估. 2014