重组白蛋白结合肽融合人白介素-11的原核可溶表达、纯化、表征及初步活性评价

Prokaryotic Soluble Expression,Purification,Characterization and Preliminary Activity Evaluation of Recombinant Albumin Binding Peptide Fused with Human Interleukin-11

目的:改善重组人白介素-11在原核表达体系中的可溶表达水平以及在体内的循环半衰期。方法:通过构建白蛋白结合肽融合人白介素-11重组蛋白表达载体[pET-30a(+)-ABD-rhIL11],实现融合蛋白在原核表达体系中的可溶表达;基于疏水层析、离子交换层析和凝胶层析建立从破菌上清液中纯化ABD-rhIL-11的方法;通过圆二色光谱及荧光发射光谱分析等鉴定ABD-rhIL-11空间结构,并进行体内外活性及药效学评价。结果:成功获得纯度高于95%的重组融合蛋白,体外结合实验结果表明,ABD-rhIL-11能够迅速与人血清白蛋白(human serum albumin,HSA)结合;细胞增殖实验结果显示,ABD-rhIL-11与对照品活性相当;药代动力学结果显示,融合蛋白能够显著延长白介素-11在大鼠体内的循环半衰期;血小板计数实验结果显示,ABD-rhIL-11能够有效刺激大鼠血小板增殖。结论:为基于白蛋白结合肽融合策略的白介素-11药物长效设计提供了新的思路。

Objective:To improve the soluble expression level of human interleukin-11 in prokaryotic expression system and enhance its circulating half-life in vivo.Methods:The recombinant protein expression vector(PBV220-ABD-rhIL11)was constructed,and it was successfully expressed in prokaryotic expression system in soluble form.Based on hydrophobic chromatography,ion exchange chromatography and gel chromatography,a method for purification of ABD-rhIL-11 from the supernatant of bacteria was successfully established.The circular dichroism spectrum and fluorescence emission spectrum analysis confirmed that the spatial structure of the purified ABD-rhIL-11 was folded correctly.In vivo and in vitro activity and pharmacodynamic evaluation was carried out.Results:The purity of recombinant fusion protein was higher than 95%.The results of in vitro binding experiments showed that ABD-rhIL-11 could rapidly bind to human serum albumin.The results of cell proliferation experiment showed that the activity of ABD-rhIL-11 was equivalent to that of the control group.Pharmacokinetic results showed that the fusion protein could significantly prolong the circulating half-life of interleukin-11 in rats.The results of platelet count test showed that ABD-rhIL-11 could effectively stimulate platelet proliferation in rats.Conclusion:The results of this study would provide a new idea for the long-term design of interleukin-11 drugs based on albumin binding peptide fusion strategy.