重组抗凝蛋白-新蛭素的原核表达研究

目的:重组新蛭素(EH)是在抗凝蛋白水蛭素的氨基末端添加3个氨基酸(EPR)的衍生物,以往EH的表达工艺沿用水蛭素的酵母表达工艺,生产周期长、目标蛋白表达效率相对较低。而水蛭素类的蛋白在大肠杆菌中往往以包涵体形式表达,后期的分离纯化收率较低,无法适应产业化。为了提高EH的生产效率,探索了EH在大肠杆菌中的可溶性表达。方法:首先通过PCR的方法获得eh的cDNA,PCR产物连接入原核表达载体pET-22或pET-24中获得重组表达质粒,将重组表达质粒转化大肠杆菌BL21(DE3)或BL21(plySs),获得重组工程菌BL21(DE3)-pET-24-eh,BL21(DE3)-pET-22-eh,BL21(plySs)-pET-22-eh。重组工程菌进行IPTG诱导,SDS-PAGE和Western blot鉴定表达产物。结果:EH在3个重组工程菌中均可实现可溶性表达。表达水平较高的为BL21(DE3)-pET-24-eh工程菌;之后通过优化诱导温度,时间,诱导剂浓度、诱导前菌种密度,确定最佳条件为:37℃,诱导6h,IPTG浓度为0.4μmol/L,诱导前菌种密度在OD₆₀₀=1左右。诱导产物经分离纯化,其纯度可达96.93%。最后通过蛋白含量测定及抗凝活性检测,确定表达的EH蛋白本身无抗凝活性,被FXa裂解后可以释放出水蛭素的抗凝活性。结论:实现了EH在大肠杆菌中的可溶性表达,表达周期短,有望提高EH的生产效率,为EH的产业化奠定了基础,也为水蛭素类产品的生产提供了新的工艺途径。

The Prokaryotic Expression of an Anti-coagulant Protein of EH

Objective: EH is a derivative of hirudin with three amino acids (EPR) at the amino terminal of hirudin. The expression of EH in yeast has long time of production and low efficiency of expression. To improve the production efficiency of EH, the soluble expression of EH in E. coli was studied. Method: Three recombinant prokaryotic expression engineering bacteria, BL21 (DE3)-pET-24-eh, BL21(DE3)-pET-22-eh, BL21(plySs)-pET-22-eh were constructed. The three engineering bacteria were cultured and EH expression was induced by IPTG. Results: The results of SDS-PAGE and Western blot showed that EH was expressed in intracellular soluble pattern in all three engineering bacteria, and the expression level is better in BL21(DE3)-pET-24-eh. Then in BL21(DE3)-pET-24-eh, the induction temperature, the induction time, the concentration of IPTG and the density of bacteria at induction time were optimized. The optimized condition is as follows: induction at 37 ℃ for 6 h, IPTG concentration is 0.4μmol/L, the bacteria density at induction time is OD₆₀₀ = 1. Finally, the specific anticoagulant activity of purified EH protein (the purity is 96.93%) and the cleavage product of EH by FXa were determined. Conclusion: The results indicated that the intact EH has no anticoagulant activity, and after cleavage with FXa, EH released anticoagulant activity of hirudin. Therefore, the prokaryotic expression system of EH, and EH were constructed which can be expressed in an intracellular soluble pattern. These results will support the subsequent fermentation process and the ultimate industrialization of EH.