重组水蛭素的原核表达及分离纯化

Prokaryotic Expression and Purification of Recombinant Hirudin

为了提高水蛭素的生产效率以适应生物医药应用的需求,试验通过对水蛭素基因进行密码子优化,构建了重组水蛭素原核表达系统,并对D600nm值、诱导时间、诱导剂IPTG浓度和诱导温度4种诱导条件进行了探索,同时通过引入试验设计(design of experiment,DOE)方案将4种因素对诱导效率的影响进行进一步系统的优化,使用His-Trap亲和层析对重组蛋白进行纯化,并通过凝血酶滴定法对重组水蛭素的活性进行测定。结果显示,试验成功构建重组水蛭素原核表达载体,水蛭素蛋白为可溶性表达。单因素变量优化后的诱导条件为:在菌体密度D600nm值为0.4时,加入1mmol/L IPTG,37℃下诱导7h,重组水蛭素蛋白表达效率最高,占菌体总蛋白66.5%;而DOE试验优化结果为:在菌体密度D600nm值为0.6时,加入0.82mmol/L IPTG,31.9℃下诱导7.6h,预测重组水蛭素蛋白表达效率最高,占菌体总蛋白72.7%,显著高于单因子变量法优化结果(P<0.05)。进一步分析发现,各影响因素之间存在明显的交互效应,影响了单因素试验结果的准确性。通过亲和层析纯化后的重组水蛭素纯度可达99%,抗凝活性为114ATU/mg。研究成功构建了重组水蛭素原核表达载体,对其表达条件进行了优化,并获得了重组水蛭素蛋白,为水蛭素应用于生物医药奠定了基础。

In order to improve the production efficiency of hirudin to meet the demand of medical application,the prokaryotic expression system of recombinant hirudin was constructed by optimizing codon of hirudin gene in this study.The four induction factors of D600 nmvalue,induction time,inducer IPTG concentration and induction temperature were optimized,and the DOE experimental scheme was designed to study the interaction of four factors.The recombinant protein was purified by His-Trap affinity chromatography and the recombinant hirudin activity was determined by thrombin titration.The results showed that the recombinant hirudin prokaryotic expression vector was successfully constructed and the expressed hirudin protein was soluble.The best induction condition acquired through single factor optimization was addition 1 mmol/L IPTG at D600 nm=0.4 and 37 ℃for 7 h,which collected the recombinant protein accounting for 66.5%of the total protein.However,the DOE experiment result suggested that the best induction condition was addition 0.82 mmol/L IPTG at D600 nm=0.6 and 31.9 ℃ for 7.6 h,the recombinant hirudin protein accounted for 72.7% of the total protein,which was significantly higher than that of the single factor method（P〈0.05）.Further analysis showed that there were obivous interaction among all four influence factors,which might affect the accuracy of the single factor experi-ment.The recombinant hirudin protein purified by affinity chromatography could achieve 99%purity,and its anticoagulant activity was 114 ATU/mg.Finally,the recombinant hirudin prokaryotic expression vector was successfully constructed,its expression condition was optimized,and the recombinant hirudin protein was obtained,which laid the foundation for the application of hirudin in medical application.