Cloning Of Two Fungal Immunomodulatory Protein Genes And Expression In Pichia Pastoris GS115

The fungal immunomodulatory proteins (FIPs) are a kind of micro-proteins extracted from macro fungi with immunomodulatory activity in recent years. The structure and biological functions of FIPs have similarity with lectins and immunoglobulin. Until now, at least eleven FIPs were found from Ganoderma lucidium, Ganoderma tsugae, Ganoderma microsporum, Ganoderma japoncium, Flammulina velutipes, Volvariella volvacea, Ganoderma sinense, Nectria haematococca, Ganoderma astum and Antrodia camphorate, they are LZ-8, FIP-gts, FIP-gmi, FIP-gja, FIP-fve, FIP-vvo, FIP-gsi, FIP-nha, FIP-gas and FIP-aca. The immunomodulatory activity of the FIPs is mainly to stimulate lymphocyte proliferation and induce the release of several cytokines and autophagy.This paper is divided into two parts. One part is that we transform the FIP-fve gene into Pichia pastoris GS115for inducible and constitutive expression to explore the best way of expression and biological activity. The FIP-fve gene and PGAP gene were cloned from the genome of Flammulina Velutipes fruiting body and P. pastoris GS115to construct inducible expression vector pPIC9-FIP-fve and constitutive expression vector pPIC9-PGAP-FIP-fve. These two vectors were transformed P. pastoris GS115, and the correction were confirmed by yeast colony PCR. Inducibe expression of rFIP-fve was observed when the P. pastoris GS115cells were cultured with1%methanol for4day, and the inducible expression level was up to158.2mg/L at the4th day. The constitutive expression level reached46.3mg/L and29.5mg/L at4th day and5th day using glucose and glycerol, respectively. The correction of rFIP-fve was proved via SDS-PAGE and Western blot. Then the rFIP-fve was purified by Ammonium sulfate precipitation and Sephadex-G-100gel chromatography. The test of biological activity showed rFIP-fve could agglutinate human red blood cells, stimulate proliferation of murine splenocytes and enhance the secretion of interleukin-2realse from murine T cells, which had a similar biological activity to the natural FIP-fve.The other part is the FIP-cve gene was cloned from the genome of Coriolus versicolor mycelia by PCR and genome walking to identify the existence of the fungal immunomodulatory protein. The peptide sequence of FIP-cve is99%and86%homologous with that the FIP-gap and LZ-8. Then we constructed the expression vector pPIC9-FIP-cve and introduced it into the P. pastoris GS115. Transformations were identified by PCR. The positive recombinant protein was induced by methanol. SDS-PAGE showed rFIP-cve was correctly expressed in the P. pastoris expression system. That would lay the foundation for the further and deeper study of FIP-cve.