重组真菌免疫调节蛋白对巨噬细胞RAW 264.7激活应答的研究

Effects of recombinant FIP on the activation of macrophages RAW 264.7

真菌免疫调节蛋白(fugal immunomodulatory proteins,FIPs)是一类从高等大型真菌中分离的、具有生物活性的小分子蛋白质。在之前的研究中,通过基因改组技术,对来自赤芝(Ganoderma lucidum)和紫芝(G.sinense)的FIP进行了重组改造,获得了具有333bp的FIP突变体FIP-SN15。本研究通过原核表达系统获得了重组FIP-SN15(rFIP-SN15),并利用荧光定量PCR(quantitative real-time PCR,qRT-PCR)技术在转录水平上对rFIP-SN15诱导巨噬细胞TNF-α的功能及其机制进行研究。结果表明,rFIP-SN15在原核表达系统中的产量约为35.31mg/L;rFIP-SN15处理细胞后,TNF-α的转录水平显著上调(P<0.05),并且Akt的转录水平上调极其显著(P<0.01)。此外,ARG1和mTOR的mRNA水平上调显著(P<0.05)。因此,rFIPSN15能够激活小鼠腹腔巨噬细胞RAW 264.7。本研究为进一步验证FIP-SN15的生物学功能提供了有益的参考。

Fugal immunomodulatory proteins（FIP）are a kind of bioactive proteins from macrofungi.In previous studies,FIP-SN15,an intragenus recombinant,was shuffled from intrageneric shuffled library constructed by FIP-glufromGanoderma lucidumand FIP-gsi fromG.sinense.In the present study,rFIPSN15 was expressed in prokaryotic expression system and the function and mechanism of rFIP-SN15 on the activation of macrophages were studied using quantitative real-time PCR（qRT-PCR）.Results showed that rFIP-SN15 was expressed with a yield of approximately 35.31 mg/L.After rFIP-SN15 treatment,the transcriptional level of TNF-αin macrophages was increased（P〈0.05）and the mRNA level of Akt was also increased（P〈0.01）.Moreover,transcriptional levels of ARG1 and mTOR were significantly increased（P〈0.05）.Therefore,rFIP-SN15 could activate mouse peritoneal macrophages RAW 264.7.This research provided a beneficial reference for the further verification of the biological function of FIP-SN15.