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目的：在原核细胞中表达具有生物活性的重组水蛭素变异物１（ｒＨＶ１）并进行重组产物的分离纯化研究。方法：以质粒ｐＢＶ２２０为载体，在大肠杆菌ＤＨ５α中表达ｒＨＶ１，发色底物法测定其抗凝血酶生物活性；利用超滤、ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－５０以及凝血酶亲合层析进行重组产物的分离纯化。结果：在大肠杆菌中获得了ｒＨＶ１的活性表达，表达量约占总蛋白的１６．９％，活力达到２０～３０ＡＴＵ／ｍｌ培养液；初步的纯化研究得到了具抗凝生物活性的ｒＨＶ１纯品，ＳＤＳ聚丙烯酰胺凝胶电泳呈单一条带。结论：重组水蛭素变异物１的活性表达及其分离纯化研究填补了国内空白，为研制国产高效重组抗凝药物带来希望。
Objective: To express bioactive hirudin mutant 1 (rHV1) in prokaryotic cells and to study the isolation and purification of recombinant hirudin. Methods: The plasmid pBV220 was used as a vector to express rHV1 in E. coli DH5α, and its antithrombin biological activity was assayed by chromogenic substrate method. The recombinant protein was characterized by ultrafiltration, DEAE-Sephadex A-50 and thrombin affinity chromatography Isolation and Purification. Results: The expression of rHV1 in Escherichia coli was about 16.9% of the total protein and its activity was 20 ~ 30ATU / ml. The purified rHV1 with anticoagulant activity SDS polyacrylamide gel electrophoresis showed a single band. CONCLUSION: The study on the activity of recombinant hirudin mutant 1 and its isolation and purification have filled the gap in our country and brought hope for the development of highly efficient recombinant anticoagulant drugs in China.