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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 闕玲玲 | |
| dc.contributor.author | Chi-Chi Wen | en |
| dc.contributor.author | 溫琦琦 | zh_TW |
| dc.date.accessioned | 2021-06-08T00:31:20Z | - |
| dc.date.copyright | 2013-07-19 | |
| dc.date.issued | 2013 | |
| dc.date.submitted | 2013-07-02 | |
| dc.identifier.citation | Addie, D.D., 2004, Feline coronavirus--that enigmatic little critter. Vet J 167, 5-6.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17692 | - |
| dc.description.abstract | 貓傳染性腹膜炎 (Feline infectious peritonitis, FIP) 為貓冠狀病毒 (Feline coronavirus , FCoV) 造成之致命性免疫媒介性疾病，至目前為止仍無有效疫苗或治療。干擾素 (Interferon, IFN) 為動物受到病毒感染時產生之細胞激素，具免疫調節或抗病毒效用，已被使用於多種人類病毒性疾病之臨床用藥。貓源干擾素 (Feline Interferon, feIFN) α, β, ω亦於體外實驗當中，被證實具有抗FCoV活性。此外，由於FIP發病貓隻體內IFN-γ表現量被發現明顯低於感染FCoV但臨床健康之貓隻，因此較高的IFN-γ之表現被推測能避免FIP發病。本研究從貓隻淋巴細胞選殖出IFN-γ基因，及合成feIFN-ω基因，透過大腸桿菌系統進行蛋白質表現，並於體外評估重組feIFN-γ與feIFN-ω蛋白質使用於FIP治療之可能性。結果顯示，重組之IFN-γH及IFN-ωH蛋白質分別於最高濃度2000 LRU/ml (Laboratory reference units/ml) 及106 LRU/ml 細胞存活率達100%，皆未偵測得任何細胞毒性。而IFN-γH 於1- 1000 LRU/ml下，抑制FCoV複製達28-40%; IFN-ωH於0.8- 105 LRU/ml下，抑制FCoV複製達94.6-99.5%。在免疫調控方面，經重組IFN-γH處理後之單核細胞與淋巴細胞，其IL-12及IFN-γ mRNA兩者表現量皆上升，綜合上述結果，重組之貓源IFN-γ及IFN-ω具有潛力成為未來臨床FIP治療藥物之一。 | zh_TW |
| dc.description.abstract | Feline infectious peritonitis (FIP) is a fetal immune-mediated disease caused by feline coronavirus (FCoV). At present, effective vaccine and treatments are still unavailable for FIP. Interferons (IFNs) are cytokines produced by the host animals upon virus infection. With either immune-modulation or antiviral effect, some IFNs have been using routinely in the clinic of human medicine. There are three feline IFN (feIFN) i.e., feIFN-α, β, ω, that shown anti-FCoV activity in vitro. As the expression level of IFN-γ appeared to be markedly depressed in FIP-cats in comparison to clinically healthy feline coronavirus-infected cats. Higher IFN-γ production has been suggested to prevent the onset of FIP. Therefore, we attempted in this study to clone feIFN-γ gene from feline lymphocytes and to synthesis feIFN-ω gene. Using an E.coli expression system the recombinant feIFN-γ and feIFN-ω were evaluated in vitro for their possibility to be used in the future FIP therapy. The result showed that 1-1000 LRU/ml (Laboratory reference units/ml) IFN-γH can inhibit FCoV replication up to 28-40%, and that 0.8-105 LRU/ml IFN-ωH can inhibit FCoV replication up to 94.6-99.5%, both IFN-γH and IFN-ωH did not produce any cytotoxicity in high concentration 2000 LRU/ml and 106 LRU/ml respectively by using cell viability assay. For the immune modulatory effect, after treating monocytes and lymphocytes by IFN-γH, the expression of IL-12 and IFN-γ mRNA were both found increased. Taking these results together, the recombinant feIFN-γ and feIFN-ω could be the potential candidates for clinical FIP therapy. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T00:31:20Z (GMT). No. of bitstreams: 1 ntu-102-R00629010-1.pdf: 1497961 bytes, checksum: c59b333ba6005f886b90f29c551b6cf2 (MD5) Previous issue date: 2013 | en |
| dc.description.tableofcontents | 中文摘要 vi
ABSTRACT vii 第一章、序言 1 第二章、文獻探討 3 第一節、貓傳染性腹膜炎 (Feline infectious peritonitis, FIP) 3 第二節、貓冠狀病毒特徵 3 一、分類 3 二、基因體特性 4 三、病毒之血清分型 5 第三節、貓冠狀病毒之感染與疾病 6 一、流行病學 6 二、致病機制 7 三、臨床症狀及診斷 8 四、治療與預防 9 第四節、干擾素 10 一、人源干擾素 (Human interferon, huIFN) 10 二、貓源干擾素 (Feline Interferon, feIFN) 11 第三章、材料與方法 12 第一節、貓源IFN-γ基因選殖 12 一、淋巴細胞培養與活化 12 二、基因萃取與純化 12 三、TA 轉殖接合反應 (TA cloning) 13 四、勝任細胞 (Competent cell) 之製備 13 五、轉型作用 (Transformation) 13 六、重組質體之複製與確認 13 第二節、IFN-γ質表現 14 一、次選殖 (Subcloning) 14 二、蛋白質誘導表現 14 三、蛋白質電泳 (SDS-page) 14 四、蛋白質之純化 15 五、蛋白質透析 15 六、蛋白質定量 15 七、西方墨點法 (Western blot) 15 八、重組IFN-γ之Trx及His tag移除 16 九、蛋白質表現系統之改良 16 第三節、重組之IFN-ω蛋白質 17 第四節、IFN生物活性測試 17 一、細胞及病毒培養與定量 17 二、IFN抗VSV之分折 18 第五節、IFN抗貓冠狀病毒之分析 18 一、細胞及病毒培養與定量 18 二、細胞活性試驗 (Cell viability) 19 三、抑制貓冠狀病毒複製試驗 19 第六節、IFN-γ免疫調節分析 20 一、單核細胞及淋巴細胞與IFN-γ作用 20 二、Real-time RT-PCR偵測單核細胞之IL-12表現量 20 三、半定量PCR偵測淋巴細胞之IFN-γ表現量 21 第七節、統計分析 21 第三章、結果 22 第一節、貓源IFN-γ基因選殖 22 第二節、IFN-γ蛋白質表現 22 第三節、IFN生物活性測試 23 第四節、IFN抗貓冠狀病毒之分析 23 一、IFN對於fcwf-4不具細胞毒性 23 二、IFN抑制FCoV之複製 24 第五節、IFN-γ免疫調節分析 24 第四章、討論 26 參考文獻 50 表1 feIFN重組蛋白質之表現與純化系統 31 表2 重組feIFN蛋白質之活性及抗FCoV效力之比較 31 圖1大腸桿菌表現載體pET-32a圖譜 32 圖2 重組IFN蛋白質序列示意圖 33 圖3 以設計之引子對進行RT-PCR增幅貓源IFN-γ基因 34 圖4 重組質體核苷酸序列與GenBank IFN-γ序列比對 35 圖5 重組質體胺基酸序列與GenBank IFN-γ序列比對 36 圖6 E.Coli AD494 (DE3) pLysS誘導表現之重組IFN-γ蛋白質 37 圖7 以西方墨點法確認純化之IFN-γ蛋白質 38 圖8 以Anti-feIFN-γ pAb進行西方墨點法確認Thrombin切割作用情形 39 圖9 IFN-γH蛋白質電泳及西方墨點法 40 圖10 IFN-γ抗VSV活性 41 圖11 IFN-ω抗VSV活性 42 圖12 IFN保護細胞對抗VSV感染之保護率 43 圖13 MTT分析不同重組IFN蛋白質對Fcwf-4細胞活性影響 44 圖14 IFN-γH預先處理細胞後FCoV攻毒之CPE情形 45 圖15 IFN-ωH預先處理細胞後FCoV攻毒之CPE情形 45 圖16 以病毒斑試驗評估IFN-γH抗病毒效力 46 圖17 以病毒斑試驗評估IFN-ωH抗病毒效力 47 圖18單核細胞IL-12 mRNA表現量 48 圖19 淋巴細胞IFN-γ mRNA表現量 49 | |
| dc.language.iso | zh-TW | |
| dc.title | 貓源干擾素之表現及其抑制貓冠狀病毒感染之評估 | zh_TW |
| dc.title | Expression and Evaluation of Feline Interferon against Feline Coronavirus Infection | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 101-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林俊宏,廖泰慶,林辰栖,林昭男,蘇璧伶 | |
| dc.subject.keyword | 貓傳染性腹膜炎,貓冠狀病毒,干擾素,細胞激素, | zh_TW |
| dc.subject.keyword | Feline infectious peritonitis,Feline coronavirus,Interferons,Cytokines, | en |
| dc.relation.page | 61 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2013-07-02 | |
| dc.contributor.author-college | 獸醫專業學院 | zh_TW |
| dc.contributor.author-dept | 獸醫學研究所 | zh_TW |
| 顯示於系所單位： | 獸醫學系 | |
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