Expression Of Ganoderma Lucidum Fungal Immunomodulatory Proteins FIP-glu And FIP-SN15 Shuffled From Two Genes Of Ganoderma Species In Saccharomyces Cerevisiae And Their Functions

Fungal immunomodulatory proteins（FIPs）are a kind of bioactive substances obtained from some edible and medicinal mushrooms.Their structures and functions are similar to immunoglobulin superfamily.They also have the immunoregulatory effects such as anti-tumor,anti-allergy and stimulating immune cells to produce various cytokines which means they have clinical application prospect and medicinal value.In this study,the full-length cDNA fragment of the fungal immunomodulatory protein from Ganoderma lucidum,designated as FIP-glu,and an intrageneric protein from G.lucidum and G.sinensis known as FIP-SN15 were used to explore the expression of FIPs in Saccharomyces cerevisiae in order to provide theoretical basis for the production of FIPs by using S.cerevisiae expression system.In this study,recombinant FIP-glu（rFIP-glu）and recombinant FIP-SN15（rFIP-SN15）were expressed in S.cerevisiae INVSc1 strain using the expression vector pYES2/NT b.Recombinant vectors pYES2-FIP-glu and pYES2-FIP-SN15 were constructed and then transformed into S.cerevisiae INVSc1.The recombinant positive clones were screened by SD/Ura^-inducing medium and identified by PCR technique.The transformants were induced by galactose,and the induced yeast cells were collected and crushed for extracting yeast intracellular proteins.Then cobalt metal affinity resin was used for purification of the rFIP-glu and rFIP-SN15.The expression levels of these two proteins reached up to 1.27 mg/L and 1.51 mg/L,respectively.After detected and analyzed by SDS-PAGE and Western blotting,we found that both of rFIP-glu and rFIP-SN15 contained glycosylation.Then purified proteins were used to evaluate the immunoreactivity on murine splenocytes at 1,2 and4μg/mL concentration.The gene expression levels of interferon-γ（IFN-γ）and interleukin-2（IL-2）in murine splenocytes were detected by real time quantitative PCR method.The results indicated that within a certain range of concentrations（1-4μg/mL）,rFIP-glu and rFIP-SN15 both could significantly increase the mRNA levels of IFN-γand IL-2 in murine splenocytes.Nevertheless,rFIP-glu was better at increasing the expression of IL-2,and rFIP-SN15 was better at increasing the expression of IFN-γ.Meanwhile,statistical optimization of media composition（carbon and nitrogen sources）and culture condition（pH）were optimized for increasing the production of FIPs.After three single factor experiments,three optimal levels of these three factors were screened,and L₉（3³）orthogonal experiments were arranged which which included 3 factors and 3 levels.After data processing,the optimal induction conditions were obtained:initial pH 5.5,20 g/L galactose concentration and 9.0 g/L nitrogen source concentration.In conclusion,the successful expression of rFIPs in S.cerevisiae indicated that a large number of rFIPs can be easily and quickly obtained by genetic engineering.They have similar structures and biological functions as those isolated from natural fungi.The strategy of functional expression of FIP-glu and FIP-SN15 gene in S.cerevisiae may lay the foundation for further exploration of the biological activity and pharmacal application of FIPs.