以嗜甲醇酵母菌表現靈芝屬免疫調節蛋白質LZ-8、GMI和GFO-1並探討其免疫功能

Overexpression of LZ-8, GMI and GFO-1, the immunomodulatory proteins from Ganoderma spp. in methylotrophic yeasts and investigation of their immune functions

賴明毅(Ming-I Lai)

指導教授 ： 黃慶璨

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.00150

摘要

由靈芝 (Ganoderma lucidum) 分離的免疫調節蛋白質LZ-8已證實具有多種免疫調節功能如促進淋巴球增生及抑制全身性過敏反應。本實驗室於2005年以genome walking方式由小孢子靈芝 (G. microsporum) 及拱狀靈芝 (G. fornicatum)釣取到LZ-8相似基因gmi、gfo-1及gfo-2 。之後發現GMI及GFO-1可刺激BALB/c小鼠骨髓樹突細胞分泌IL-12、刺激小鼠巨噬細胞株J774A-1分泌TNF-α以及刺激人類淋巴癌T細胞株Jurkat分泌IL-2。
　　由於前人研究多以細胞株進行免疫調節活性測試，測定的細胞激素種類也較少。故本研究欲以嗜甲醇酵母菌表現LZ-8、GMI、GFO-1及GFO-2，並以小鼠初代細胞探討其免疫調節功能，作為往後進行動物實驗之參考。
　　研究結果顯示，以搖瓶培養並經五倍濃縮，在誘導前菌體細胞濃度可達1.44 ± 0.03 × 1010 cfu/mL (rLZ-8 / H. polymorpha)、2.94 ± 0.23 × 109 cfu/mL (rGMI / P. pastoris) 及8.60 ± 0.14 × 109 cfu/mL (rGFO-1 / H. polymorpha)。以1%甲醇誘導1天後再濃縮純化，可得到23.3 ± 0.1 mg/L rLZ-8、4.3 ± 0.4 mg/L rGMI及8.5 ± 0.2 mg/L rGFO-1。在免疫調節活性部分，rLZ-8、rGMI及rGFO-1均可在未以其他物質刺激的條件下活化免疫系統，亦可刺激小鼠腹腔細胞及脾臟細胞產生細胞激素，具有抑制過敏反應及抑制腫瘤的潛力。與 rLZ-8及rGFO-1相比，rGMI在刺激IL-1β、IL-2、 IFN-γ、IL-4及IL-5的效果有明顯差異，可能與N端的α helix以及前35個胺基酸序列的差異性有關，未來可以點突變探討胺基酸序列的差異對上述細胞激素表現的影響。rGFO-2無法以嗜甲醇酵母菌順利表現，可能因本身基因序列具有高GC比例，密碼子使用率，或是與第60個胺基酸附近的loop序列有關，未來可設計引子修改密碼子，或針對loop的glycine進行點突變。

關鍵字

真菌免疫調節蛋白質 ； 免疫調節功能 ； 嗜甲醇酵母菌

並列摘要

LZ-8, a fungal immunomodulatory protein (FIP) isolated from Ganoderma lucidum exhibits potent mitogenic effect upon human peripheral blood lymphocytes (PBL) and prevents the production of systemic anaphylaxis reaction in mice. Three lz-8-like genes, gmi, gfo-1 and gfo-2 were successfully isolated from G. microsporum and G. fornicatum previously in our laboratory. GMI and GFO-1 were found to stimulate bone marrow derivated dendritic cells from BALB/c mice, murine macrophage J774A-1 and Jurkat T cells to secret IL-12, TNF-α and IL-2, respectively.
From previous studies, the immune functions of FIPs were estimated based on cell line model, and only a few cytokines were determined. In this study, LZ-8, GMI, GFO-1 and GFO-2 were expressed in methylotrophic yeasts and their immune functions were assayed in BALB/c mice primary cells.
The average production of rLZ-8 and rGFO-1 in H. polymorpha were 23.3 ± 0.1 and 8.5 ± 0.2 mg/L, respectively, while rGMI reached 4.3 ± 0.4 mg/L in P. pastoris. All the rLZ-8, rGMI and rGFO-1 were found to activate the immune system spontaneously and to stimulate BALB/c mice peritoneal cells and splenocytes to secret several cytokines such as IL-1β, TNF-α, IL-6, IL-2, IFN-γ, IL-4 and IL-5. Suggesting that these recombinant proteins exist the potential in suppressing allergic disease and anti-tumor effect. For the stimulation of IL-1β, IL-2, IFN-γ, IL-4 and IL-5, rGMI was significantly different from rLZ-8 and rGFO-1 probably due to the difference in amino acid sequence of N-terminal α helix region. The unsuccessful expression of gfo-2 in methylotrophic yeasts might be explained by the high GC content, different codon usage and the glycine-rich loop.

並列關鍵字

Fungal immunomodulatory proteins ； immune modulation

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