重组鼠肝素辅因子Ⅱ在大肠杆菌的表达及纯化

RECOMBINANT MURINE HEPARIN COFACTORⅡ:EXPRESSlON IN E. COLI AND PURIFICATION

重组鼠肝素辅因子Ⅱ（ｒＨＣⅡ）ｃＤＮＡ在ＰＥＴ一５ａ质粒载体被构建，克隆到大肠杆菌株ＤＨ５ａ；ｒＨＣⅡ蛋白在大肠杆菌ＢＬ２１（ＤＥ３）被表达为非糖基化蛋白。用肝素琼脂糖柱，ＭｏｎｏＱ和ＭｏｎｏＳ柱层析纯化ｒＨＣⅡ，获得高活性和纯度单一的ｒＨＣⅡ。ｒＨＣⅡ在免疫反应及硫酸皮肤素或肝素依赖性凝血酶抑制活性方面，与天然鼠血浆ＨＣⅡ无明显差异。

iuse heparin cofactorⅡ(HC Ⅱ)cDNA clone was constituted and recombined inPET-5; plasmid vector system and cloned in E. coli-DH5a. Recombinant mouse HC Ⅱ pro-tein was expressed in E. coli-BL21 (DE3)as a non-glycosylated protein. Expressed HC Ⅱwas pirified from E. coli by three-step procedure that included heparin-sepharose column,Mono Q and MonoS column. The final product has thrombin inhibitory activity which is de-pendmt on the present of dermatan sulfate or heparin and displays a single band on SDS-PAGE. The recombinant mouse HC Ⅱ shares a similar behavior both in functional activityand in immune reaction with mouse plasma HCⅡ .