Identification a novel Ganoderma FIP gene from Ganoderma capense and its functional expression in Pichia pastoris.

Abstract

Ganoderma capense is a precious medicinal fungus in China. In this study, a novel fungal immunomodulatory protein gene, named as FIP-gca, was cloned from G. capense by homologous cloning. Sequencing analysis indicated that FIP-gca was composed of 336bp, which encoded a polypeptide of 110 amino acids. Protein sequence blasting and phylogenetic analysis showed that FIP-gca shared homology with other Ganoderma FIPs. FIP-gca was effectively expressed in Pichia pastoris GS115 at an expression level of 166.8mg/L and purified using HisTrap™ fast-flow prepack columns. The immunomodulation capacity of rFIP-gca was demonstrated by that rFIP-gca could obviously stimulate cell proliferation and increase IL-2 secretion of murine spleen lymphocytes. Besides, antitumor activity of rFIP-gca towards human stomach cancer AGS cell line was evaluated in vitro. Cell wound scratch assay proved that rFIP-gca could inhibit migration of AGS cells. And flow cytometry assay revealed that rFIP-gca could significantly induce apoptosis of AGS cells. rFIP-gca was able to induce 18.12% and 22.29% cell apoptosis at 0.3μM and 0.6μM, respectively. Conclusively, the novel FIP-gca gene from G. capense has been functionally expressed in Pichia and rFIP-gca exhibited ideal immunomodulation and anti-tumour activities, which implies its potential application and study in future.