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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。
The purpose of this study was to obtain a recombinant vWF cleavage protease (ADAMTS13) and to further investigate its role in thrombosis and hemostasis. The recombinant plasmid pSecTag-ADAMTS13 encoding the full-length ADAMTS13 was transfected into Hela cells by lipofectamine. The positive clone cell lines were screened with Hygromycin-B and expanded to obtain the supernatant. The purified protein was purified by Ni-NTA agarose column and gradient imidazole leaching method. Purity and immunological activity of the purified product were identified by SDS-PAGE and Western blotting. Protein-shearing activity was determined by GST-His double-antibody sandwich method. The results showed that a cell strain ADAMTS2-4 capable of constitutively secreting recombinant ADAMTS13 protein was successfully obtained, and 5.8 mg of recombinant protein was purified per 1L of culture supernatant. Western blotting showed that ADAMTS13 showed a single band at 190 kDa and the protein had a shear activity of 6.4 U / mL (ADUTS13 activity was 1 U / ml in normal human mixed plasma). The recombinant protein has good immunogenic activity and enzyme activity, which laid a good foundation for further study on the mechanism and application of ADAMTS13.