Expression And Biological Activity Of Intergeneric Recombinant Fungal Immunomodulatory Protein(FIP-SJ75)

Fungal immunomodulatory proteins(FIPs)are a class of biologically active small molecule proteins,which are derived from edible and medicinal fungi with high sequence and structural similarity.FIPs have a wide range of biological activities,such as anti-tumor,anti-allergy,promoting immune cell proliferation,and regulating cytokines.As people’s interest in the study of their medicinal properties continues to increase,it is important to establish a method for efficiently expressing soluble FIPs and clearly studying the underlying mechanisms of their biological activities.FIP-SJ75,a novel fungal immunomodulatory protein gene,was shuffled from the genes of three different mushroom species: Ganoderma lucidum,Flammulina velutipes,and Volvariella volvacea,with FIP-glu,FIP-fve,FIP-vvo,were used as the objects in this study.Based on the prokaryotic expression vectors,both pCold TF::FIP-SJ75,pCold TF::FIP-glu,pCold TF::FIP-fve,and pCold TF::FIP-vvo were successfully constructed.Soluble recombinant FIP-SJ75(rFIP-SJ75),rFIP-glu,rFIP-fve,rFIP-vvo were efficiently expressed in Escherichia coli Rosetta cells by IPTG induction;rFIPs were confirmed by SDS-PAGE and Western Blot,respectively;the expression level of rFIPs was determined by the Bradford method.The results showed that the above rFIPs were correctly expressed in E.coli,and the yields were 17.69 mg/L,15.37 mg/L,13.42 mg/L,and11.88 mg/L,respectively.After the His tag and the Trigger Factor chaperone were excised by thrombin,the purified rFIP-SJ75 was identified by LC/Q-TOF MS.The results showed that the amino acid sequence matching rate of the predicted 6 peptides after splicing was 84.3%,indicating that the recombinant protein with a molecular weight of about13 k D after expression and purification was the expected rFIP-SJ75.In order to further explore and compare the direct anti-tumor activity and immunomodulatory activity of rFIP-SJ75 with three other parental rFIPs,human hepatocellular carcinoma cell HepG2 and mouse peritoneal macrophage RAW264.7 were selected as materials for bioactivity experiments in vitro.CCK-8 cytotoxicity test of HepG2 hepatocellular carcinoma cells showed that the cytotoxicity of rFIP-SJ75 on HepG2 cells was dose-dependent,and when the cells were treated with 8 μg/m L of rFIP-SJ75,the cytotoxicity was stabilized.The cytotoxicity comparison of the four rFIPs showed that when the protein concentration was 32 μg/m L,the inhibitory effect of rFIP-SJ75 on HepG2 proliferation was significantly stronger than that of rFIP-vvo.CCK-8 cytotoxicity test results of RAW264.7 macrophages showed that rFIP-SJ75 significantly promoted the proliferation of RAW264.7 cells at the low protein concentration(1 ～ 8μg/m L).Neutral red intake experiment to investigate the effect of rFIPs on phagocytosis of RAW264.7 cells showed that rFIP-SJ75 potentiated the phagocytosis of RAW264.7 cells in a dose-dependent manner in the concentration range of 1 ～ 16 μg/m L.When treated with 8 μg/m L rFIPs,rFIPs significantly enhanced the phagocytosis of macrophages,and rFIP-SJ75 enhanced the phagocytosis of macrophages better than rFIP-glu and rFIP-fve.Finally,the effects of rFIPs on macrophage inflammation-related gene expression were investigated by RT-q PCR.The results showed that after treatment of RAW264.7 cells with four rFIPs for6 h,8 μg/m L of rFIPs could promote the expression of two pro-inflammatory related genes(TNF-α and IL-6)and inhibit the two anti-inflammatory related genes(Expression of IL-10 and TGF-β1).Among the four rFIPs,as for pro-inflammatory related genes,rFIP-SJ75 promoted the expression of TNF-α less than rFIP-fve;it promoted the expression of IL-6 more strongly than rFIP-glu and rFIP-vvo.For anti-inflammatory related genes,rFIP-SJ75 inhibited the expression of IL-10 strongly than rFIP-fve and rFIP-vvo;it inhibited the expression of TGF-β1 more strongly than rFIP-vvo.In conclusion,the novel intergeneric recombinant fungal immunoregulatory protein rFIP-SJ75 was successfully expressed in E.coli.Within a certain concentration range,rFIP-SJ75 has a significant direct anti-tumor effect on human hepatocellular carcinoma cell HepG2.Within a certain concentration range,rFIP-SJ75 can significantly promote the proliferation of mouse peritoneal macrophage RAW264.7,enhance the phagocytic ability of RAW264.7 cells,promote the expression of pro-inflammatory related genes and inhibit the expression of anti-inflammatory related genes.This study provided a theoretical basis for the subsequent research and industrial production of FIP-SJ75 as an immunomodulator for some inflammation-related diseases.