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目的制备具有生物学活性的重组人巨噬细胞移动抑制因子(hMIF),为进一步研究MIF的功能和筛选MIF阻断剂奠定基础。方法构建PET11b-MIF、PET32a-MIF表达载体,建立重组MIF纯化方案,通过体内、体外实验对不同表达方式的重组hMIF互变异构酶活性、促进炎症因子释放,加重脓毒症动物死亡率的作用进行对比研究。结果成功表达了两种重组hMIF,建立并优化了重组MIF的纯化方法,发现PET11b-MIF表达的的MIF经SP Sepharose FF阳离子交换层析和Hitrap 26/60sepbacryl S-300分子筛纯化后具有明显的互变异构酶的活性,能剂量依赖性地刺激RAW264.7细胞释放NO和TNF-α、IL-6,并加重了脓毒症动物的死亡率。而PET32a-MIF表达的N端带有Trx标签的hMIF(Trx-hMIF)无上述作用。结论我们成功制备了具有生物学活性的MIF,为深入研究MIF的功能和筛选MIF阻断剂奠定了基础。
Objective To prepare a biologically active recombinant human macrophage migration inhibitory factor (hMIF), which will lay the foundation for the further study on the function of MIF and the screening of MIF blockers. METHODS: PET11b-MIF and PET32a-MIF expression vectors were constructed and a recombinant MIF purification protocol was established. Through in vitro and in vivo experiments, recombinant hMIF tautomerase activity of different expression patterns was promoted, the release of inflammatory cytokines was promoted, and the mortality rate of septic animals was increased The role of comparative study. Results Two recombinant hMIFs were successfully expressed and purified. The results showed that MIF expressed by PET11b-MIF was significantly cleaved by SP Sepharose FF cation exchange chromatography and Hitrap 26 / 60sepbacryl S-300 molecular sieve Mutant enzyme activity, can dose-dependently stimulate RAW264.7 cells release NO and TNF-α, IL-6, and aggravated the mortality of septic animals. The Trx-tagged hMIF (Trx-hMIF) at the N-terminus of PET32a-MIF did not. Conclusions We successfully prepared biologically active MIF and laid the foundation for further study on the function of MIF and the screening of MIF blockers.