Regulation Of Fungi Immunomodulatory Protein Expression

Fungal Immunomodulatory Protein (FIP), extracted from higherbasidiomycetes, is a kind of small molecule protein with extensivebiological functions, including anti-tumor and anti-allergy, stimulatingimmune cells to produce a variety of cytokines, etc.FIP-SN15, a novel gene shuffled from the genes of Ganodermasinensis and Ganoderma lucidum FIP, with FIP-glu, were used as theobjects in this study. Based on the construction of prokaryotic expressionvectors, both pET30a-FIP-glu and pET30a-FIP-SN15were expressed inEscherichia coli. The results showed that the recombinant proteinFIP-SN15and FIP-glu could be expressed in E. coli, the yield of whichwas35.95mg/L and36.67mg/L respectively. Analysis using BandScan V5.0software showed that the yield of recombinant FIP-SN15and FIP-gluaccounted for51.4%and72.2%of the total proteins of E. colirespectively, and the purity of reFIPs reached above90%aftermetal-affinity purification. The recombinant protein FIP-SN15consistedof111amino acids, and4peptides were identified by Q-tof MS with matching coverage rate of91.9%, and the secondary and tertiary structureof FIP-SN15were also predicted by bioinformatics method, whichsuggest that FIP-SN15is a new member in FIPs family.The anti-tumor bioactivity of recombinant protein FIP-SN15wasinvestigated by using the MTT cytotoxicity assay on four human cancercell lines, including Lung carcinoma (A549), Colon cancer (HT-29),Hepatocellular carcinoma (HCC-LM3) and Breast adenocarcinoma(MDA-MB-231) cell lines. The results demonstrated that FIP-SN15couldinhibit the cell proliferation of A549and MDA-MB-231in lowconcentrations and the IC50respectively, while the FIP-SN15could barely inhibit the cellproliferation of HCC-LM3and HT-29.A cell aggregation assay was performed with human glioblastoma(astrocytoma) cell line U-251MG. It was found that cells treated withFIP-SN15(10μg/ml) or FIP-glu (10μg/ml) started to agglutinate at thesixth hour and detached by48hour. FACS (fluorescence activated cellsorting-SN15and FIP-glu couldslightly induce apoptosis in U-251MG cells, and the totalapoptotic rateswere increased by6.03%and21.93%respectively. The results of reFIPson U-251MG cell cycle suggested that reFIPs could inhibit the G1phaseto S phase transition of U-251MG cells so as to retard the cell cycleprogression. Moreover, in this thesis, eukaryotic expression vectors of FIP-glu andFIP-gsi gene designated as PV2+-FIP-glu-HyB and PV2+-FIP-gsi-HyBwere successfully constructed.In this study, we attempted to construct both prokaryotic andeukaryotic expression vectors for FIPs, and express FIPs from differentsources in E. coli, the expression level obtained was higher than similarstudies previously reported. The agglutinating activity of reFIPs was alsotested on tumor cells, and FACS results showed that reFIPs couldeffectively inhibit the proliferation of tumor cells. The results obtainedhave laid the foundation for further development of reFIPs as the rapeuticproducts and research on the anti-tumor mechanisms of FIPs.