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  • 學位論文

貓源干擾素之表現及其抑制貓冠狀病毒感染之評估

Expression and Evaluation of Feline Interferon against Feline Coronavirus Infection

指導教授 : 闕玲玲
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摘要


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。

並列摘要


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.

參考文獻


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