重组水蛭素的原核表达及分离纯化

doi:10.16431/j.cnki.1671-7236.2018.01.001

Abstract

Abstract:

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 D_{600 nm} value, 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 D_{600 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 D_{600 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 experiment. 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.

Key words: hirudin; prokaryotic expression; induction condition optimization; DOE; affinity chromatography

CLC Number:

HUANG Kongwei, LI Zhipeng, CUI Kuiqing, SHI Deshun, LIU Qingyou. Prokaryotic Expression and Purification of Recombinant Hirudin[J]. , 2018, 45(1): 1-13.

References

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Prokaryotic Expression and Purification of Recombinant Hirudin

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