The Expressions And The Functional Characteristics Of The Mutants Of Fungal Immunomodulatory Proteins

Fungal immunomodulatory proteins(FIPs)are a series of bioactive small-molecule proteins that have been reported to modulate immune systems,inhibiting tumor growth,promote the proliferation of lymphocytes and the agglomeration of erythrocytes,and induce the secretion of cytokines in immunocytes.Our research group has obtained new recombinant genes using the DNA shuffling technology: FIP-SN15 and FIP-SJ.The former that was shuffling from FIP-glu and FIP-gsi and the latter was shuffling from FIP-glu,FIP-fve and FIP-vvo.On the basis of these achievements,we constructed prokaryotic expression vectors,pET-30a::FIP-SN15 and pET-30a::FIP-SJ,then transformed them into Escherichia coli BL21 competence cells.Further,the recombinant FIP-SN15(rFIP-SN15)and rFIP-SJ were obtained with IPTG induction expression.After that,the proteins purification processed with Ni-NTA chelation column,ultrafiltration device and Sephadex G-75 dextran gel column.In order to explore their immunomodulatory effects on macrophages,rFIP-SN15 and rFIP-SJ were treated mouse abdominal macrophages(RAW 264.7).The results of SDS-PAGE and Western blotting experiments showed that FIP-SN15 and FIP-SJ were successfully expressed in E.coli BL21 cells,and the calculation with BandScan V5 showed that the expression yield was 35.31 mg/L.The results of SDS-PAGE and Western blotting analyses demostrated that the expression yield of FIP-SN15 reached its peak after 5 h under 0.5 mM IPTG inducement,and FIP-SJ formed into inclusion bodies in the cell sediments ender 0.5 mM IPTG inducement.The protein purification results showed that the eluent with 25 mM imidazole could eliminate proteins impuririties effectively under different imidazole concentrations,and the eluent with 250 mM imidazole can purify rFIP-SN15 completely.In order to abtain elution curve,we carried Sephadex G-75 dextran gel column chromatography experiment.The protein concentration reached its peak at the eluting peak.Relatively high FIP-SN15 concentration could be obtained in approximately 1/3 column volume eluate.Mouse abdominal macrophages(RAW 264.7)were treated with different concentrations of rFIP-SN15(2 mg/L,4 mg/L,8 mg/L and 16 mg/L)and extracted total RNA.The expression level of tumor necrosis factor(TNF-?)was measured with qRT-PCR and the results showed that the TNF-? mRNA expressions level of macrophages of rFIP-SN15 increased following different concentration and showed a dose-related pattern compared with the PBS control group.The relative TNF-? expression yield of 4 mg/L rFIP-SN15 group was the highest.The protein kinase B(Akt),the mammalian target of rapamycin(mTOR),the phosphatase,tensin homolog deleted on chromosome ten(PTEN)and the arginase 1(ARG1)are related proteins on the biosynthesis pathway of TNF-?.The evaluation of the relative mRNA expression level of the related genes in the TNF-? biosynthesis pathway showed that,the increase of mRNA expression level of ARG1 and MTOR were significant(p < 0.05).The increase of Akt expression level was extremely significant(p < 0.01).These results suggested that rFIP-SN15 modulated the immunologic function by activating Akt,ARG1 and mTOR expression level to affect TNF-?.This study involved in vitro recombinant expression,purification and functions of intrageneric recombinant FIPs.In the research,it is the first time to report the mutants of FIPs.In addition,by investigating on the expression levels of parts of the related genes on the TNF-? biosynthesis pathway,the study preliminary clarify the mechanism of TNF-? inducement by rFIP-SN15 in macrophages.