Bioinformatics And Biological Activity Of Immunomodulatory Proteins In Hypsizygus Marmoreus

Hypsizygus marmoreus,a popular edible and medicinal fungus in Asian,has significant development potential and good market prospects.Fungal immunomodulatory proteins(FIPs)isolated from fungi are a class of bioactive proteins with a structure similar to immunoglobulins,which have biological activities such as anti-tumor,anti-inflammation,anti-allergy,and cytokine expression promotion.Exploring new FIPs and their medicinal value provides fresh research ideas for the development of nutritional supplements and novel drugs that take the advantage of macrofungi.In this study,the immunomodulatory protein c13717 from Ganoderma australe was used as a bait.Three novel proteins with similarity over 50% to c13717 were found through BLAST in the genome of Hypsizygus marmoreus sequenced in the laboratory,which were named FIP-hma1,FIP-hma2 and FIP-hma3,respectively.Bioinformatics analysis showed that FIP-hmas were hydrophobic proteins composed of 143-147 amino acids with a molecular weight of 14.79-15.43 k Da.The N-terminal region displayed that all three proteins had signal peptides,and they were found to be secreted extracellularly after subcellular localization.FIP-hmas contained a Cerato-platain(CP)conservative domain and belonged to the CP type FIP.Phylogenetic tree analysis indicated that FIP-hmas belonged to a new branch of the FIPs family,showing great evolutionary differences from other FIPs,which might be derived from different ancestors.The 3D structure forecasted that FIP-hmas included short α-helical,six β-folds,and many random coils,which was consistent with the conservative domain analysis that classified them as a CP type FIP.Real time fluorescence quantitative PCR was used to detect the gene expression levels of three FIP-hmas genes during the development of Hypsizygus marmoreus.The results showed that the expression levels of FIP-hma1 and FIP-hma2 were higher in the vegetative growth stages than in the reproductive growth stages,especially in the post mature stage of the mycelium.However,the expression level of FIP-hma3 was at a top level at the beginning of the vegetative growth stages and the end of the reproductive growth stages,which meant that the expression level was high when the mycelium reached half of the cultivation bottle and was at the maturity stage of the fruiting body.In order to improve the production of immunomodulatory protein of Hypsizygus marmoreus,the expression vector p Smart I-FIP-hmas was further constructed and transferred into the expression system of E.coli BL21(DE3)to express the recombinant proteins.Finally,FIP hma2 was successfully expressed and purified.The expression and purification conditions of the highly effective soluble fusion protein of the recombinant strain were screened.The best expression condition was determined to be 15 ℃,1.0 m M IPTG induction and culture for 16 hours,then the most soluble protein could be obtained.Through Ni NTA separation and gradient elution with imidazole solution,purified proteins could be collected.Using SUMO protease cleavage to remove the label,the recombinant protein FIP-hma2(r FIP-hma2)verified by SDS-PAGE and Western blot was successfully isolated and purified.The immune activity of r FIP-hma2 in vitro was studied.Western blot analysis demonstrated that r FIP-hma2 significantly improved the expression levels of i NOS,IL-1β,and IL-6 in the RAW 264.7 macrophage cells.Enzyme linked immunosorbent assay(ELISA)showed that r FIP-hma2 enhanced the release of IL-6,TNF-α,IL-1β to the extracellular fluid,indicating that r FIP-hma2 played a role in the immune response activation by activating core immune regulatory factors.In summary,this study identified three novel immunoregulatory proteins and identified their bioinformatics characteristics in Hypsizygus marmoreus.FIP-hma2 was expressed and purified in E.coli system,and the conditions of recombinant expression and purification were optimized,which provided an effective method for the recombinant production of FIPs.In vitro immune activity tests found that r FIP-hma2 increased the release of specific cytokines from macrophages,which exhibited immune activation regulatory activity.This study provided a basis for the physiological function research and industrial application of FIPs.