血管性血友病因子裂解酶CUB区的稳定表达及其鉴定

目的 该研究旨在得到血管性血友病因子裂解酶ADAMTS13中重组的CUB区蛋白,进一步研究其生物功能.方法 利用脂质体将编码CUB区序列的重组质粒pSecTag-CUB转染Hela细胞.用潮霉素（hygromycin-B）筛选得到阳性克隆细胞株,并扩大培养,收集上清.利用Ni-NTA琼脂糖柱,梯度咪唑淋洗法纯化蛋白,SDS-PAGE和Western blotting鉴定纯化产品纯度和免疫学活性.结果 成功获得一株能恒定分泌重组CUB区蛋白的细胞株CUB-7,每1 L培养上清可纯化剑6.0mg重组蛋白.Western blotting结果显示,6×His抗体能与重组蛋白在41kD处显单一条带.结论 重组蛋白具有较好的免疫原活性和纯度,为进一步研究CUB区在ADAMTS13中的作用机理和运用奠定了良好的基础.

Stable Expression and Characterization of the CUB Domain in von Willebrand Factor Cleaving Protease

Objective This study was to acquire recombinant protein of CUB domain in von Willebrand factor cleaving protease（ADAMTSl3）,for further studies on its biological function. Method We transfected the Hela cells with the plasmid pSecTag-CUB by lipofectamine.A positive cell cloning was selected by hygromycin-B. The recombinant protein was purified with Ni-NTA agarose column by gradient imidazole.The purity and immune activity of purified products were identified with SDS-PAGE and Western blotting respectively. Result We successfully constructed Hela cells CUB-7 which expressed high level of the rCUB.We received about 6.0mg recombinant protein in culture supernantants per liter purified with Ni-NTA column.The protein formed a main lane at the position of 41kD with SDS-PAGE and reacted with monoclonal antibody against 6 x His by Western blotting. Conclusion The rCUB protein had high purity, immune activity, which could establish the experimental foundation for further research on biological function and mechanism of this domain in ADAMTSlY