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

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Western blotting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1 L培养上清可纯化得到5.8 mg重组蛋白。Western blotting结果显示，ADAMTS13多抗能与重组蛋白在190 kDa处显单一条带，并且蛋白具有6.4 U/mL的剪切活性 (每毫升正常人混合血浆中ADAMTS13活性为1 U)。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。

Abstract:

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.