靈芝屬免疫調節蛋白 GMI 與 GFO-1 基因之選殖與 Pichia pastoris 之異源表現

Abstract

Ganoderma lucidum has been used in Chinese medical application for a long time. Several studies reported that G. lucidum exhibited anti-tumor and immunomodulatroy activities. Polysaccharides and triterperoid were regarded as the major bioactive substances. Another bioactive material was not found until the small protein LZ-8 was purified from mycelium of G. lucidum in 1989. In this study, LZ-8 primers were used to amplify ten strains of Ganoderma spp. genomic DNA. LZ-8-like sequences were universally found in Ganoderma spp. strains. These LZ-8-like sequences were not exactly identical to LZ-8. Some strains contained two different LZ-8-like sequences. Three new genes, gmi, gfo-1 and gfo-2, were successfully cloned from G. microsporum and G. fornicatum. Three genes, lz-8, gmi and gfo-1, were expressed extracellularly in Pichia pastoris KM71 with methanol induction. No glycosylation in recombinant proteins rePLZ, reGMI and reGFO-1 was found after MALDI-TOF analysis. The average productivities could achieve 300 mg/l culture. Proteolysis occurred at late stage of expression because of oxygen and H2O2 stress induced by methanol. Proteases could not be removed after purification but could be inhibited by EDTA and pepstatin A completely. The differences in protein conformation between rePLZ, reGMI and reGFO-1 was attributed to their amino acid sequences. reGFO-1 was conformationally similar to rePLZ while reGMI was different from rePLZ. Bone marrow derivative dendritic cells from BALB/c mice were stimulated by rePLZ, reGMI and reGFO-1 to secrete IL-12. The concentration of IL-12 stimulated by reGMI was five times higher than that by rePLZ. rePLZ, reGMI and reGFO-1 stimulated murine macrophage J774A.1 cells to secrete TNF-α and Jurkate cells（human T cell line）to secrete IL-2.