Screening, Functional Characterization Of Recombinant Fungal Immunomodulatory Proteins

Fungal immunomodulatory proteins (FIPs) is a small molecule protein with immune regulating activity. Till now, six kinds of fips gene have been isolated from Ganoderma lucidum, G. tsugae, Flammulina velutipes, G. sinensis, G. japoncium, and G. microsporum, respectively. Four FIPs, LZ-8 (FIP-glu), FIP-gts, FIP-fve, and FIP-vvo, were isolated directly from the fruit-bodies or mycelia of natural mushrooms. FIPs has widely immune regulating activity, such as mitogenicity, regulate cell cycle and induce apoptosis, induce expression of cytokine and anti-allergic effects, etc.In this study, a DNA shuffling technique was used to create two shuffled FIP protein libraries: an intrageneric group containing products of shuffling between fip-glu (fip gene isolated from Ganoderma lucidum) and fip-gsi (fip gene isolated from Ganoderma sinense) genes and an intergeneric group containing the products of shuffling between fip-glu, fip-fve (fip gene isolated from Flammulina velutipes) and fip-vvo (fip gene isolated from Volvariella volvacea) genes. The gene shuffling generated 426 and 412 recombinant clones, respectively. Using colony blot analysis, we selected clones that expressed relatively high levels of shuffled gene products recognized by specific polyclonal antibodies. We analyzed the DNA sequences of the selected shuffled genes, and explored reFIPs bioactivities of agglutinating blood cells and inducing cytokine production in splenocytes of Kunming mice in vitro. Meanwhile, the relationships between protein structure and the hemagglutination activity and between the changed nucleotide sites and expression levels were explored by bioinformatic analysis. These combined analyses identified the nucleotide changes involved in regulating the expression levels and hemagglutination activities of the FIPs. Both of the recombinant FIPs could induce interleukin (IL)-2, IL-3, IL-4, interferon (IFN)-γ, IL-2 receptor (IL-2R), lymphotoxin (LT) genes transcriptional expression.Therefore, we were able to generate recombinant FIPs with improved biological activities and expression levels by DNA shuffling, a way for generating novel therapeutic proteins and for their structural and functional studies.