臺灣博碩士論文加值系統

鴨產蛋下降綜合症病毒（DEDSV）是一種從中國鴨場分離出新興致病的黃病毒，台灣目前並未發現此病。鴨子感染DEDSV後主要導致嚴重的產蛋下降綜合症，其臨床症狀，包括厭食，腹瀉，共濟失調，癱瘓，然而最重要的是產蛋下降，導致了巨大的經濟損失。因此，開發一種方法和途徑以對抗DEDSV是當務之急。而現在已有減毒疫苗被報導是可以提供保護，但我們的目標是希望可以更經濟地製造與開發次單位疫苗。目前我們已經合成DEDSV的E基因，並將Domain I及 Domain II (DI+II)轉殖到pET32a載體中，以原核表達系統的大腸桿菌BL-21表現出E基因的domain I與domain II的蛋白。我們將表現出來的蛋白當作疫苗，評估其抗原性且進行鴨子的免疫劑量試驗。從ELISA的試驗結果，我們發現100μg/mL的免疫劑量所誘發的抗體力價是最高的。現在我們繼續以100μg/mL的免疫劑量搭配水包油包水佐劑進行動物試驗來觀察其免疫反應是否更佳，然而經過1個月的動物試驗後發現疫苗是能夠誘發體液性免疫反應，且免疫組的抗體力價在28天的最高，明顯高於對照組，綜合以上結果，以大腸桿菌表現之重組蛋白DI+II是具有發展為DEDSV次單位疫苗之潛力。

Duck egg drop syndrome virus (DEDSV) is an emerging pathogenic flavivirus isolated from ducks in China. Currently, the disease is not found in Taiwan. The infected ducks manifest clinical signs that include anorexia, diarrhea, ataxia, paralysis, and most importantly, a heavy drop in egg products that results in huge economic losses. Thus, the discovery of ways and means to combat DEDSV is urgent. While a live vaccine has been reported to provide protection .We aim to develop a subunit vaccine that may be more economical to manufacture. At present, we have synthesized the E gene, and domain I and domain II (DI+II) of E gene were cloned into pET32a vector for prokaryotic expression in Escherichia coli BL-21 as a subunit vaccine. We also have evaluated the antigenicity of vaccine and tested immunizing dose. From the results of ELISA, we found that 100μg/mL of vaccine induced highest antibody titer. Now, we have combined the vaccine with water-in-oil-in-water adjuvant and tested on ducks to observe whether the adjuvant can improve immune response. After 1 month animal test, we get the result of antibody titer. The result showed that vaccine group can induce humoral immune response and the antibody titer was significantly higher than the control group (p < 0.05) after 28 dpi. Base on the result, the E.coli expressed DI+II protein may be potential for developing an effective subunit vaccine against DEDSV.

目 錄
摘要………………………………………………………………………Ⅰ
Abstract…………………………………………………………………...Ⅲ
謝誌………………………………………………………………………Ⅴ
目錄………………………………………………………………………Ⅵ
圖表目錄…………………………………………………………………XI
第1章 緒言………………………………………………………………1
第2章 文獻回顧…………………………………………………………3
2.1 目前鴨產業現況……………………………………………………..3
2.2 鴨產蛋下降症病毒對家禽產業之影響……………………………..4
2.3 鴨產蛋下降綜合症病毒的分類與特性……………………………..5
2.4 鴨產蛋下降綜合症病毒蛋白質結構………………………………..5
2.5 鴨產蛋下降綜合症病毒之傳播途徑………………………………..6
2.6 鴨產蛋下降綜合症病毒之臨床症狀………………………………..6
2.7 鴨產蛋下降綜合症病毒之流行病學………………………………..6
2.8 疫苗開發現況………………………………………………………..7
2.9 疫苗的種類與優缺點………………………………………………..8
2.10 禽類疫苗現況……………………………………………………..10
2.11 佐劑的種類及使用………………………………………………..11
第3章 材料與方法………………………………………………………14
3.1 實驗所使用之載體、病毒基因片段及勝任細胞…………………14
3.1.1 pET32a 載體…………………………………………………..14
3.1.2 Escherichia coli DH5α…………………………………………14
3.1.3 Escherichia coli BL-21(DE3)………………………………….14
3.1.4 DEDSV之E醣蛋白……………………………………………14
3.2 DEDSV之E醣蛋白基因片段轉殖與定序……………………….15
3.2.1 DEDSV之E蛋白基因片段篩選………………………………15
3.2.2 設計E蛋白基因含限制酶切位之特異性引子………………15
3.2.3 E蛋白基因之擴增-PCR………………………………………15
3.2.4 膠體電泳法確認PCR產物大小………………………………15
3.2.5 執行膠體純化回收產物……………………………………….15
3.2.6 pET32a載體的製備……………………………………………16
3.2.7 E醣蛋白基因的製備…………………………………………..16
3.2.8 DEDSV之E gene和Domain I+II與pET32a載體之接合反應(ligation)…………………………………………………………………..16
3.2.9 轉型作用至Escherichia coli DH5α (Transformation)………..17
3.2.10 小量質體DNA萃取…………………………………………17
3.2.11 以PCR確認pET/E及pET/D I+II……………………………18
3.2.12 核酸序列定序與分析………………………………………...18
3.3 以大腸桿菌表現DEDSV E醣蛋白(含Full E及Domain I+II)…18
3.3.1 轉型作用至Escherichia coli BL-21(Transformation)…………18
3.3.2 DEDSV E醣蛋白(含Full E及Domain I+II)的表現………18
3.4重組E醣蛋白之蛋白質分析…………………………………………19
3.4.1 蛋白質電泳樣品製備………………………………………….19
3.4.2 聚丙烯醯胺膠體的製備(SDS PAGE)…………………………19
3.4.3 重組蛋白Domain I+II之定量…………………………………19
3.4.4 重組蛋白Domain I+II之純化…………………………………20
3.4.5 重組蛋白Domain I+II之抗原性分析-西方墨點法(Western blotting)…………………………………………………………………...20
3.5 疫苗之製備…………………………………………………………20
3.5.1 重組蛋白之不活化…………………………………………….20
3.5.2 疫苗調配……………………………………………………….21
3.5.3 無菌試驗……………………………………………………….21
3.6 動物試驗及免疫評估………………………………………………21
3.6.1 鴨隻試驗1-疫苗劑量之評估(Dose dependent)………………21
3.6.1.1 鴨隻試驗2-抗體力價之評估…………………………….22
3.6.1.2 酵素連結免疫吸附法(ELISA)……………………………22
3.6.2 免疫細胞增生試驗…………………………………………….22
3.6.2.1 周邊血單核球細胞(PBMC)之抽取………………………22
3.6.2.2 細胞計數…………………………………………………..23
3.6.2.3 抗原刺激…………………………………………………..23
3.6.2.4 MTT分析…………………………………………………23
3.6.2.5 細胞增生指數計算………………………………………..23
3.7.1 利用Real-time PCR檢測周邊血液細胞激素的產生量………24
3.7.1.1 PBMC刺激及RNA萃取…………………………………24
3.7.1.2 反轉錄聚合酶連鎖反應…………………………………..24
3.7.1.3 即時聚合酶連鎖反應( Real-time polymerase chain reaction )…………………………………………………………………..25
第4章 結果…………………………………………………………….26
4.1 鴨產蛋下降症病毒E基因之擴增…………………………………26
4.2 質體DNA轉型作用之確認………………………………………..26
4.3 鴨產蛋下降症病毒E基因之定序與胺基酸比對…………………26
4.4 重組蛋白(E protein)的表現與分析………………………………...26
4.5 重組蛋白(DI+II)的純化……………………………………………27
4.6 重組蛋白Domain I+II之抗原性分析……………………………..27
4.7 重組蛋白Domain I+II之定量( BSA standard)……………………27
4.8 疫苗劑量之評估……………………………………………………27
4.9 抗體力價之評估……………………………………………………28
4.10 T細胞增生試驗之結果( MTT assay)…………………………….28
4.11 細胞激素增生試驗之結果( Cytokines assay)…………………….28
第5章 討論…………………………………………………………….48
參考文獻………………………………………………………………….53
附錄……………………………………………………………………….59
Appendix 1. The structural, antigenic of envelope proteins …………59
Appendix 2. The phylogeny of representative flavivirus with DEDSV, TMUV, and STWV. ………………………………………………………60
Appendix 3. The DEDSV strain from report ………………………….61
作者簡介………………………………………………………………….62

余祁暐、劉依蓁、林彥宏。全球動物保健產業發展現況與趨勢。農業生技產業季刊。生物科技產業研究中心，2014。

陳建翰、李宜映。動物疫苗產業發展趨勢與人才職能需求研析。行政院農委會農政與農情期刊，103年6月(第264期)。

李龍湖。疫苗發展技術與實驗。動物基因轉殖與疫苗發展技術教學中心，2003。

余祁暐。臺灣動物用疫苗產業發展現況。農業生技產業季刊。生物科技產業研究中心，2013。

張德生。大腸桿菌中重組蛋白質表現之研究。博士論文。國立清華大學，2002。

田浪、馮曉聲、郭吉餘、錢雪橋。禽坦布蘇病毒研究進展。中國畜牧獸醫。2016, 43(2): 553~558

呂傳位、李寧、張振東、牛玉娟、劉思當。鴨坦布蘇病毒對育成鴨和產蛋鴨致病性對比研究。山東農業大學動物科技學院，2012。

余磊、閆大為、高旭元、肖亞莉、劉紹瓊、李雪松、李國新、李澤君。鴨坦布蘇病毒E蛋白結構域III原核表達產物誘導中和抗體的研究。中國農業科學院上海獸醫研究所，2015。

姬希文、閆麗萍、顏丕熙、李國新、張七斤、李澤君。鴨坦布蘇病毒抗體間接ELISA檢測方法的建立。中國農業科學院上海獸醫研究所，2011。

Liu Z, Ji Y, Huang X, Fu Y, Wei J, Cai X, et al. An adapted duck Tembusu virus induces systemic infection and mediates antibody-dependent disease severity in mice. Virus Res. 2013;176:216-22.

Li G, Gao X, Xiao Y, Liu S, Peng S, Li X, et al. Development of a live attenuated vaccine candidate against duck Tembusu viral disease. Virology. 2014;450-451:233-42.

Fu Y, Ji Y, Liu B, Dafallah RM, Zhu Q. Development of a solid-phase competition ELISA to detect antibodies against newly emerged Duck Tembusu virus. J Virol Methods. 2015;224:73-6.

Zhao D, Huang X, Liu Y, Han K, Zhang J, Yang J, et al. Domain I and II from newly emerging goose tembusu virus envelope protein functions as a dominant-negative inhibitor of virus infectivity. Res Vet Sci. 2015;98:121-6.

Ti J, Zhang L, Li Z, Zhao D, Zhang Y, Li F, et al. Effect of age and inoculation route on the infection of duck Tembusu virus in Goslings. Vet Microbiol. 2015;181:190-7.

Zhang Y, Li X, Chen H, Ti J, Yang G, Zhang L, et al. Evidence of possible vertical transmission of Tembusu virus in ducks. Vet Microbiol. 2015;179:149-54.

Dai L, Li Z, Tao P. Evolutionary analysis of Tembusu virus: evidence for the emergence of a dominant genotype. Infect Genet Evol. 2015;32:124-9.

Zhou H, Yan B, Chen S, Wang M, Jia R, Cheng A. Evolutionary characterization of Tembusu virus infection through identification of codon usage patterns. Infect Genet Evol. 2015;35:27-33.

Harakuni T, Andoh K, Sakamoto RI, Tamaki Y, Miyata T, Uefuji H, et al. Fiber knob domain lacking the shaft sequence but fused to a coiled coil is a candidate subunit vaccine against egg-drop syndrome. Vaccine. 2016.

Yan P, Zhao Y, Zhang X, Xu D, Dai X, Teng Q, et al. An infectious disease of ducks caused by a newly emerged Tembusu virus strain in mainland China. Virology. 2011;417:1-8.

Li S, Zhang L, Wang Y, Wang S, Sun H, Su W, et al. An infectious full-length cDNA clone of duck Tembusu virus, a newly emerging flavivirus causing duck egg drop syndrome in China. Virus Res. 2013;171:238-41.

Ma T, Liu Y, Cheng J, Liu Y, Fan W, Cheng Z, et al. Liposomes containing recombinant E protein vaccine against duck Tembusu virus in ducks. Vaccine. 2016;34:2157-63.

Cha SY, Kang M, Moon OK, Park CK, Jang HK. Respiratory disease due to current egg drop syndrome virus in Pekin ducks. Vet Microbiol. 2013;165:305-11.

Sun XY, Diao YX, Wang J, Liu X, Lu AL, Zhang L, et al. Tembusu virus infection in Cherry Valley ducks: the effect of age at infection. Vet Microbiol. 2014;168:16-24.

Chen P, Liu J, Jiang Y, Zhao Y, Li Q, Wu L, et al. The vaccine efficacy of recombinant duck enteritis virus expressing secreted E with or without PrM proteins of duck tembusu virus. Vaccine. 2014;32:5271-7.

Zhao, D., Huang, X., Han, K., Liu, Y., Yang, J., Liu, Q., An, F., Li, Y., 2015. Protective immune response against newly emerging goose tembusu virus infection induced by immunization with a recombinant envelope protein. Lett Appl Microbiol 61, 318-324.

Sun, L., Li, Y.X., Zhang, Y., Han, Z.X., Xu, Y., Kong, X.G., Liu, S.W., 2014. Adaptation and Attenuation of Duck Tembusu Virus Strain Du/CH/LSD/110128 following Serial Passage in Chicken Embryos. Clin Vaccine Immunol 21, 1046-1053.

Fu Y, Ji Y, Liu B, Dafallah RM, Zhu Q. Development of a solid-phase competition ELISA to detect antibodies against newly emerged Duck Tembusu virus. J Virol Methods. 2015;224:73-6.

Lu Y, Dou Y, Ti J, Wang A, Cheng B, Zhang X, et al. The effect of Tembusu virus infection in different week-old Cherry Valley breeding ducks. Vet Microbiol. 2016;192:167-74.

Yu K, Sheng ZZ, Huang B, Ma X, Li Y, Yuan X, Qin Z, Wang D, Chakravarty S, Li F, Song M, and Sun H. Structural, antigenic, and evolutionary characterizations of the envelope protein of newly emerging Duck Tembusu Virus. PloS one 8: e71319, 2013.

Schneeweiss A, Chabierski S, Salomo M, Delaroque N, Al-Robaiy S, Grunwald T, Burki K, Liebert UG, and Ulbert S. A DNA vaccine encoding the E protein of West Nile virus is protective and can be boosted by recombinant domain DIII. Vaccine 29: 6352-6357, 2011.

Su J, Li S, Hu X, Yu X, Wang Y, Liu P, et al. Duck egg-drop syndrome caused by BYD virus, a new Tembusu-related flavivirus. PLoS One. 2011;6:e18106.

Martina BE, Koraka P, van den Doel P, van Amerongen G, Rimmelzwaan GF, and Osterhaus AD. Immunization with West Nile virus envelope domain III protects mice against lethal infection with homologous and heterologous virus. Vaccine 26: 153-157, 2008.

Beasley DWC, and Barrett ADT. Identification of Neutralizing Epitopes within Structural Domain III of the West Nile Virus Envelope Protein. Journal of Virology 76: 13097-13100, 2002.
Cao Z, Zhang C, Liu Y, Liu Y, Ye W, Han J, Ma G, Zhang D, Xu F, Gao X, Tang Y, Shi S, Wan C, Zhang C, He B, Yang M, Lu X, Huang Y, Diao Y, Ma X, and Zhang D. Tembusu virus in ducks, china. Emerging infectious diseases 17: 1873-1875, 2011.

Deng YQ, Dai JX, Ji GH, Jiang T, Wang HJ, Yang HO, Tan WL, Liu R, Yu M, Ge BX, Zhu QY, Qin ED, Guo YJ, and Qin CF. A broadly flavivirus cross-neutralizing monoclonal antibody that recognizes a novel epitope within the fusion loop of E protein. PloS one 6: e16059, 2011.

Volk DE, May FJ, Gandham SH, Anderson A, Von Lindern JJ, Beasley DW, et al. Structure of yellow fever virus envelope protein domain III. Virology. 2009;394:12-8.

Chavez JH, Silva JR, Amarilla AA, Moraes Figueiredo LT. Domain III peptides from flavivirus envelope protein are useful antigens for serologic diagnosis and targets for immunization. Biologicals. 2010;38:613-8.

Oliveira ERA, Mohana-Borges R, de Alencastro RB, Horta BAC. The flavivirus capsid protein: Structure, function and perspectives towards drug design. Virus Research. 2017;227:115-23.

Niu H-m, Huang X-m, Han K-k, Liu Y-z, Zhao D-m, Zhang J-f, et al. Development of Double Antibody Sandwich ELISA for Detection of Duck or Goose Flavivirus. Journal of Integrative Agriculture. 2013;12:1638-43.

Chu JHJ, Chiang CCS, Ng ML. Immunization of Flavivirus West Nile Recombinant Envelope Domain III Protein Induced Specific Immune Response and Protection against West Nile Virus Infection. The Journal of Immunology. 2007;178:2699-705.

Wu X, Shi Y, Yan D, Li X, Yan P, Gao X, et al. Development of a PCR-Based Reverse Genetics System for an Attenuated Duck Tembusu Virus Strain. PLoS One. 2016;11:e0156579.

Thontiravong A, Ninvilai P, Tunterak W, Nonthabenjawan N, Chaiyavong S, Angkabkingkaew K, et al. Tembusu-Related Flavivirus in Ducks, Thailand. Emerg Infect Dis. 2015;21:2164-7.

Shaozhou W, Li C, Zhang Q, Meng R, Gao Y, Liu H, et al. Duck tembusu virus and its envelope protein induce programmed cell death. Virus Genes. 2015;51:39-44.

Li N, Wang Y, Li R, Liu J, Zhang J, Cai Y, et al. Immune responses of ducks infected with duck Tembusu virus. Front Microbiol. 2015;6:425.

Dai L, Li Z, Tao P. Evolutionary analysis of Tembusu virus: evidence for the emergence of a dominant genotype. Infect Genet Evol. 2015;32:124-9.

Dai L, Song J, Lu X, Deng YQ, Musyoki AM, Cheng H, et al. Structures of the Zika Virus Envelope Protein and Its Complex with a Flavivirus Broadly Protective Antibody. Cell Host Microbe. 2016;19:696-704.

Ershova AS, Gra OA, Lyaschuk AM, Grunina TM, Tkachuk AP, Bartov MS, et al. Recombinant domains III of Tick-Borne Encephalitis Virus envelope protein in combination with dextran and CpGs induce immune response and partial protectiveness against TBE virus infection in mice. BMC Infect Dis. 2016;16:544.