血管性血友病因子裂解蛋白酶的稳定表达及其活性检测  

Stable expression and characterization of the von Willebrand factor cleaving protease

作　　者：马珍妮[1] 董宁征[1] 张敬宇[1] 苏健[1] 汪安友[1] 阮长耿[1] 

机构地区：[1]苏州大学附属第一医院江苏省血液研究所卫生部血栓与止血重点实验室,苏州215006

出　　处：《生物工程学报》2010年第2期244-248,共5页Chinese Journal of Biotechnology

基　　金：国家自然科学基金项目(No.30670904)资助~~

摘　　要：本研究旨在得到重组的血管性血友病因子裂解蛋白酶(ADAMTS13),进一步研究其在血栓止血中的作用。利用脂质体将编码ADAMTS13全长序列的重组质粒pSecTag-ADAMTS13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Westernblotting鉴定纯化产品纯度和免疫学活性,采用GST-His双抗夹心法测定蛋白剪切活性。结果显示,成功获得一株能恒定分泌重组ADAMTS13蛋白的细胞株ADAMTS2-4,每1L培养上清可纯化得到5.8mg重组蛋白。Western blotting结果显示,ADAMTS13多抗能与重组蛋白在190kDa处显单一条带,并且蛋白具有6.4U/mL的剪切活性(每毫升正常人混合血浆中ADAMTS13活性为1U)。重组蛋白具有较好的免疫原活性和酶活性,为进一步研究ADAMTS13作用机理和运用奠定了良好的基础。This study was to acquire recombinant protein of von Willebrand factor cleaving protease （ADAMTS13, a disintegrin and metalloprotease with a thromboSpondin type 1 motifs 13）, for further studies on its biological function in thrombosis and hemostasis. We transfected the Hela cells with the plasmid pSecTag-ADAMTS13 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. We also measured the enzymatic activity of recombinant protein （rADAMTS13） by GST-His two-site ELISA assay. The results showed that we successfully constructed Hela cells ADAMTS2-4 which expressed high level of rADAMTS13. We received about 5.8 mg recombinant protein in culture supernantants per liter purified with Ni-NTA column. The protein formed a main lane at the position of 190 kDa with SDS-PAGE and reacted with polyclonal antibody against ADAMTS13 by Western blotting. The amount of rADAMTS13 activity was 6.4 U/mL, according to the normal plasma defined as 1 U/mL. In conclusion, rADAMTS13 protein had high purity, immune activity and good enzymatic activity, which could establish the experimental foundation for further research on biological function and mechanism of this unique metalloprotease.

关 键 词：血管性血友病因子裂解蛋白酶 真核表达 重组蛋白 

分 类 号：R554.1[医药卫生—血液循环系统疾病]