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

• Main Authors: Ming-I Lai, 賴明毅
• Other Authors: 黃慶璨
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/12638399437234960986

碩士 === 國立臺灣大學 === 微生物與生化學研究所 === 98 === 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.