| 摘要: | 摘要 干擾素 ( interferon ) 屬於細胞激素的成員之一，具重要免疫調節( immunomodulation ) 功能，會因病毒感染細胞而被誘發生成，並抑制人體內病毒的複製與細胞生長，是抵抗多種病毒感染的第一道防線。干擾素-γ (interferon-gamma, IFN-γ) 是可抑制腫瘤及參與免疫反應，其免疫調節能力比其他干擾素為佳，也是重要的醫療蛋白之一。本實驗的設計(1) 希望能利用RhPV蚜蟲病毒 (Rhopalosiphum padi virus) 5’UTR 的IRES序列及水母 ( Aequorea victoria) 的綠螢光 (enhanced green flouresecent protein) 蛋白當作偵測標誌，以cap-independent的轉譯機制來表現綠螢光以快速挑選重組病毒。(2)將IFN-γ基因3’末端同義融合 (in frame fusion) 六個組胺酸 ( histidine) 序列的核酸序列，以利用固定化金屬親和性層析法 (Immobilized metal affinity chromotagraphy, IMAC ) 純化出IFN-γ蛋白。(3) 利用對醣蛋白 ( glycoprotein ) 上醣基具有專一性作用的植物凝集素 ( lectin ) 來分析三種不同的昆蟲細胞株，包括秋行軍蟲 ( Spodoptera frugiperda , SF21 )、甜菜夜蛾 ( Spodotera exigua, SE ) 與斜紋夜蛾 ( Spodoptera litura, SL ) 所生產純化的IFN-γ蛋白醣基化不同。(4) 利用抗登革熱病毒試驗作為測試對無血清培養基所生產經IMAC純化的IFN-γ蛋白 ( purify SF21) 以及有血清、無血清培養基所生產的IFN-γ蛋白， 我們分定義為 (serum SF21 and no serum SF21) 與市售 ( commercial ) 的IFN-γ蛋白比較生物活性的不同。結果顯示，確實可利用RhPV-IRES-EGFP中帶有綠螢光可快速挑選病毒，經IMAC所純化IFN-γ蛋白的純度更高達96％以上，自SE細胞株所生產純化出的IFN-γ蛋白擁有更多protein band與較多mannose的醣基化形式，所以其醣基化的形式較SF21的細胞株更複雜。而從SL的細胞株所生產的IFN-γ蛋白則與SF21生產的差不多，最後，由無血清培養基所生產IFN-γ蛋白的濃度達 301.1 pg/ml，可有效地抑制 90％登革熱病毒的生成。而由無血清陪養基所生產後經IMAC純化IFN-γ蛋白的濃度達 389.47 pg/ml，也可有效地抑制 90％登革熱病毒的生成。 Abtract Interferon plays a role in inhibition of tumor growth and participates in immunoreactions. Among interferons, interferon-γ was one of the most important therapeutic protein and ability of immunodulation of interferon-γ was better than other type interferon. The purpose of the thesis discussed how to produce bioactive human interferon-γ rapidly. (Ⅰ) Use RhPV (Rhopalosiphum padi virus) 5’UTR IRES (internal ribosome entry site) and green fluorescent protein to develop a rapid screening strategy for recombinant virus . (Ⅱ) Use hexahistidine peptide coding sequence introduced at 3’ end of the recombinant interferon-γ gene, thus after expressed in baculovirus expression system, the secreted interferon-γ protein in cultured supernatants was purified by IMAC (immunobilized metal affinity chromatography). (Ⅲ) Use lectin, which was specific to the terminal of the carbohydrates, to analyze glycosylated pattern of purified interferon-γ produced by three different insect cells (included SF21, SL, and SE cells). (Ⅳ) Use anti-dengue virus activity to test biological activity of interferon-γ produced from two different cultured conditions, which was serum and serum-free medium and interferon-γ produced by serum-free cultured condition was purified by IMAC. In this study, we confirmed the RhPV 5’UTR IRES sequence coupled with green fluorescence could facilitate the isolation of recombinant virus. And the purity of interferon-γ protein purified by IMAC reached 96％. Furthermore, purified interferon-γ protein produced from the SE cell had more glycosylated types of protein and mammose bands, thus the glycosylated types were more complex than the SF21 cell. Besides, the glycosylated types of the interferon-γ protein from the SL cell is similar to the SF21 cell. In the end, the concentration of the interferon-γ protein produced by serum-free cultured condition reaches 301.1 pg/ml and it could inhibit 90％ dengue virus productions. In addition, the concentration of the purified interferon-γ protein under serum-free cultured condition reaches 389.47 pg/ml and it could also inhibit 90％ dengue virus productions. |
