牛干擾素α重組蛋白之抗病毒效力與免疫功能分析

干擾素（Interferon, IFN）最早於1957年被發現，為一種具有強烈抗病毒能力的可溶性蛋白。根據干擾素功能性與細胞表面受體構型不同，可將其分為三型，即第一型、第二型與第三型干擾素。其中，第一型干擾素主要由干擾素α與干擾素β所組成，所能達到抑制病毒複製的程度最為顯著，其功用包含抑制病毒複製與介導生物訊號之旁分泌，藉由該途徑可警惕鄰近細胞誘導干擾素刺激基因（Interferon-stimulated gene, ISG）的上調，使未受感染之細胞形成抗病毒狀態，以達到限制病毒傳播之目的。直至今日，干擾素α已廣泛用於乙型肝炎、丙型肝炎與人類免疫缺陷病毒的偕同治療，且達到顯著降低病毒載量的功效。本研究將牛干擾素α基因嵌入載體中並通過大腸桿菌表現系統（Escherichia coli expression system）進行表現，重組牛干擾素α蛋白續透過IPTG（Isopropyl β-D-1-thiogalactopyranoside）誘導及鎳離子純化樹脂（Nickel-NTA chelating resin）純化，續以SDS-PAGE及西方墨點法進行分析。此外，通過在牛腎細胞（Madin-Darby Bovine Kidney, MDBK）中進行牛干擾素之體外試驗（In vitro assay），以測定重組蛋白之最適處理時間與劑量，並評估其抗病毒效力。最後在體內試驗（In vivo assay）通過牛隻肌肉注射重組蛋白，並以即時定量PCR檢測動物體之細胞激素水平差異。結果顯示，重組牛干擾素α蛋白已被成功表現與純化。在體外試驗結果中，利用重組蛋白處理的組別與陽性對照組相比，病毒核酸量有顯著下降的趨勢，且大部分ISGs在24小時之內皆提升。同時在體內試驗結果中，牛隻免疫後一日之周邊血液單核球中的IFN-γ水平高於對照組達6.5倍。綜上研究結果，可用於進一步建構牛抗病毒劑之研究。

關鍵字

牛干擾素α ；大腸桿菌表現系統；重組蛋白；抗病毒效力

並列摘要

Interferon (IFN), initially discovered in 1957, is a soluble glycoprotein with strong antiviral effects. Based on functional properties and cell surface receptors configuration, interferons could divide into three types, which are type I, type II and type III interferons. Among the type I interferons, IFN-α and IFN-β had the most significant effects, which include the inhibition of viral replication, and the mediation of the paracrine biological signaling process. Through this process, the neighboring cells were alerted of the viral infection causing the interferon-stimulated genes (ISGs) to be up-regulated and the uninfected cells to enter the antiviral state, thus limiting further viral transmission. Interferon-α is currently being used for hepatitis B, hepatitis C and human immunodeficiency virus treatments achieving significant reduction in viral load. In this study, the bovine interferon-α (BoIFN-α) gene was inserted into a vector for an Escherichia coli expression system. The bovine interferon-α recombinant protein (rBoIFN-α) will be induced using Isopropyl β-D-1-thiogalactopyranoside (IPTG) and purified by Nickel-NTA chelating resin, then analyzed by SDS-PAGE and Western blot. In addition, in vitro assays in Madin-Darby Bovine Kidney cells（MDBK）were performed to determine the optimal dose and treatment time and evaluate the antiviral efficacy. Lastly, in vivo assay was conducted in cattle treated intramuscularly with the rBoIFN-α, and subsequently tested for differences in expression of cytokines by real-time PCR. The result shows that rBoIFN-α had been expressed and purified successfully. The in vitro assays showed that compared to the positive control, the viral nucleic acids of the groups which treated with rBoIFN-α had a significant decline, and most of ISGs had increased within 24 hours. Meanwhile, the IFN-γ in peripheral blood mononuclear spheres (PBMC) 1 day after immunization is about 6.5 times higher than control in the in vivo assay. The results of this study could be applied in further research on the construction of bovine antiviral agent.