重组水蛭素克隆的表达及其产物的分离纯化

Expression and purification of recombinant hirudin

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 the recombinant hirudin variant 1 (rHV1) with biological activity in prokaryotic cells and then isolate and purify the expressed products.Methods:The hirudin variant 1 gene in plasmid vector pBV220 was expressed in E.coli strain DH5α.The biological activity of rHV1 was determined with chromogenic substrate method.The expressed product was purified by ultrafiltration,DEAESephadex A50 filtration and thrombinSepharose 4B affinity chromatography.Results:The expressed rHV1 accounted for approximately 16.9% of E. coli cell proteins with an activity of 20～30 ATU (antithrombin unit) per milliliter culture. The purified rHV1 showed a homogeneous band on SDSPAGE.Conclusions:This is the first report in China on successful expression of hirudin variant 1 gene in E.coli and purification of the expressed rHV1.