Isolation And Purification Of Novel Hirudins From The Leech Hirudinaria Manillensis

Cardiovascular Disease severely impair humanity’s health, "The Reported of Chinese Cardiovascular Disease in 2010" points out:approximately 3,000,000 people died of the cardiovascular disease every year in our country, accounts for completely 40% of the whole world’s death. The correlation data demonstrated:this kind of disease’s caused by the non-normal hemoglutination process, with the blood vessel wall, the blood acorn tube, the antifreeze, the filament dissolves and blood changing and so on. In this paper, we focused our attention on the isolation of hirudin-like polypeptides from Hirudinaria manillensis, a species which is significantly more specialized for mammalian parasitism than Hinido medicinalis, expected to gain an anticoagulant that more suitable for cardiovascular patients.The technology of ultrafiltrate and lyophillization were carried on the concentration, unified the DEAE anion column hromatography and the ODS opposition column hromatography on the separation. As the result, we separated four kind of proteins initially, identified by the matrix auxiliary laser analysis-flight time second-level mass spectrum, and establishment exclusive database.In the extraction process, we compared with different precipitation method by the indicator of anticoagulant recovery. Thus, we determined to adopt the ethanol precipitation, and used the single factor to optimal the extraction process. The final result was:added the cold ethanol in the supernate to the final density was 20%, elimination foreign protein by centrifugalization, the supernate continues to add cold ethanol until the density was up to 80%. After centrifuge,we gained the crude anticoagulant matter.In purification aspect, we compared with different chromatographic media by the indicator of anticoagulant recovery and protein’s purity. Thus, we determined to adopt the anion-exchange chromatography and reversed-phase chromatography. After inspects, the best separation condition of anion-exchange chromatography was:buffer phosphate as equilibration buffer, sodium chloride solution with different density were taken as the elution buffer solution,254nm online monitor, assayed the thrombin inhibition simultaneously; The best separation condition of reversed-phase chromatography was: 20%EtOH-H2O-TFA(contained 0.03% NaH2PO4) as equilibration buffer,30%EtOH-H2O-TFA(contained 0.03% NaH2PO4) as the elution buffer solution,254nm online monitor, assayed the thrombin inhibition too. Finally,we gained four four kind of proteins,one of them performed remarkable, had a specific activity.The pure polypeptide was shown to be homogenous by HPLC and sodium dodecyl sulphate polyacrylamide gel electrophoresis, with an apparent molecular mass of about 19200 daltons under reducing conditions.In the content’s determination, we used the thrombin to determine its biological activity, BCA protein assay kit to determine its protein content, polyacrylamide gel electrophoresis to trace protein and MALDI-TOF-MS/MS to identify the proteins.The polypeptide we got had a specific activity of about 2742ATU/mg. Contrasted its peptide mass fingerprintin with the data which gathers in the database——National Center for Biotechnology Information, none similar proteins were found.The results show that using chilled alcohol precipitation method, anion-exchange chromatography and C18 reversed-phase chromatography to isolate anticoagulant polypeptides from Hirudinaria manillensis, the technology is reasonable, stable and feasible.