開發貓泛白血球減少症之干擾素抗病毒治療劑

干擾素( Interferon, IFN) 為細胞激素的一種，有抗病毒的功效，且具有物種專一性。本研究選用的干擾素-阿法( Interferon alpha, IFNα )為Ｉ型，可由淋巴細胞、巨噬細胞、內皮細胞等多種細胞分泌。貓泛白血球減少症病毒 ( Feline panleukopenia virus, FPV) 又稱貓小病毒，為小病毒科 (parvoviridae)，小病毒屬(parvovirus)之無封套單股DNA病毒( ssDNA)。FPV會造成貓發燒、嘔吐下痢、病理學檢查可見嚴重的泛白血球減少症。本研究欲將貓源IFNα應用於貓泛白血球減少症( Feline panleukopenia, FPL) 作為抗病毒之治療劑。利用重組載體pET24a (+)與貓IFNα序列合成質體後經轉形至勝任細胞DH5α內，並進一步提取質體，送入表現系統內表現重組貓源干擾素蛋白質。再經Ni-NTA樹酯純化，透析置換緩衝溶液中的鹽類後收取IFNα蛋白。利用SDS-PAGE確認蛋白質純化效果並以西方墨點法確認純化後蛋白質帶有6x His標籤；並以貓腎細胞株(Crandell Rees Feline Kidney, CRFK) 培養出貓小病毒進行體外試驗(in vitro)，並分別對於重組貓源干擾素α的劑量及作用時間進行測試。試驗干擾素濃度0.05μg/μl能成功抑制病毒增殖2至4天，達到短效治療病毒感染病症的效果。

關鍵字

貓泛白血球減少症病毒；干擾素；抗病毒治療劑

並列摘要

Interferons (IFN) are a kind of cytokine that has antiviral effects and has species specificity. The Interferon alpha (IFNα) selected in this study is type I, which can be secreted by lymphocytes, macrophages, endothelial cells, and other cells. Feline panleukopenia virus (FPV) is an unenveloped single-stranded DNA virus (ssDNA) of the family Parvoviridae and the genus Parvovirus. FPV can cause fever, vomiting and diarrhea in cats, and severe panleukopenia can be seen on pathological examination. This study intends to apply feline IFNα to feline panleukopenia (FPL) as an antiviral therapeutic agent. The recombinant vector pET24a (+) and cat IFNα sequence were used to synthesize the plasmids that were used to transform DH5α competent cells. Successful transformants were selected by antibiotic screening. The plasmids were further extracted and transform to the expression system for protein expression. After purification by Ni-NTA resin, the IFNα protein is recovered after dialysis to replace the salts in the buffer solution. Using SDS-PAGE and Western blotting method, the purity of the harvested protein and the presence of the 6X His tag after purification was confirmed, respectively. Feline Panleukopenia Virus (FPV) was cultured with cat kidney cell line (Crandell Rees Feline Kidney, CRFK) for in vitro experiments (in vitro), and the dose and duration of interferon were tested. The test interferon concentration of 0.05μg/μl can successfully inhibit the proliferation of the virus for 2 to 4 days, and achieve the effect of short-acting treatment of viral infections.