Establishment of an Antiviral Activity Assay and the Identification and Partial Purification of Interferon-like Protein from Rainbow Trout Gonadal Cells (RTG-2)

抗病毒活性檢測法之建立與虹鱒生殖腺細胞株（RTG-2）之抗病毒蛋白質的辨識與部份純化

林世斌(Shih-Bin Lin) ； 林志信(Chih-Hsin Lin) ； 徐亞莉(Ya-Li Hsu)

《Zoological Studies》 40卷3期 (2001/07) Pp. 240-246

摘要

本實驗建立了一套快速（24小時內）有效的抗病毒活性生物檢測法。低濃度感染性胰臟壞死病毒（IPNV）（MOI介於0.00064至0.4）在虹鱒生殖腺細胞株（RTG-2）內培養超過12小時後，可明顯提高RTG-2細胞在後續的高濃度（MOI=100）IPNV病毒攻毒下的存活率。IPNV病毒可誘發RTG-2產生抗病毒蛋白質。實驗亦發現誘發液中殘留的病毒可造成受測樣本之抗病毒活性高估或假顯性的現象。若經濃縮之抗病毒蛋白質粗誘發培養液，分別利用HPLC分子篩色層分析管柱及nativePAGE作部份純化。前者的分析圖譜中發現兩處（100及18 kDa）有較高的抗病毒活性，惟不明顯。而後者（native PAGE）則在約100 kDa處發現有明顯抗病毒活性，且經西方墨點法之抗人類干擾素γ多株抗體處理後，發現該處有訊號產生。本實驗亦發現經由十日齡細胞所誘發出來的抗病毒活性，高於由六日齡細胞所誘發出來者約1.6至3.2倍。

關鍵字

類干擾素 ； 抗病毒蛋白質 ； 虹彩鱒魚生殖腺細胞株 ； 感染性肝胰臟壞死病毒

並列摘要

In this study, a rapid (performed within 24 h) and effective bioassay method free from overestimation or false positives for antiviral activity due to virus residue (within an MOI range of 6.4 × 10^(-4) to 0.4) was established. Antiviral proteins, generated by infectious pancreatic necrosis virus (IPNV) induction of rainbow trout gonadal cells (RTG-2), were partially purified by HPLC size exclusion chromatography and native polyacrylamide gel electrophoresis (PAGE); the chromatography demonstrated 2 possible antiviral proteins with molecular weights of 100 and 18 kDa, respectively. Electrophoresis produced 1 antiviral protein with a molecular weight of about 100 kDa identified through Western blotting with polyclonal anti-human IFN-γ. The yield of antiviral protein obtained from 10-d-old cells was 1.6-3.2 times higher than that from 6-d-old cells.

並列關鍵字

Interferon-like protein ； Antiviral protein ； RTG-2 ； IPNV