血管性血友病因子裂解蛋白酶的穩(wěn)定表達及其活性檢測

馬珍妮，董寧征，張敬宇，蘇健，汪安友，阮長耿

蘇州大學附屬第一醫(yī)院 江蘇省血液研究所 衛(wèi)生部血栓與止血重點實驗室，蘇州 215006

血管性血友病因子裂解蛋白酶的穩(wěn)定表達及其活性檢測

馬珍妮，董寧征，張敬宇，蘇健，汪安友，阮長耿

蘇州大學附屬第一醫(yī)院 江蘇省血液研究所 衛(wèi)生部血栓與止血重點實驗室，蘇州 215006

本研究旨在得到重組的血管性血友病因子裂解蛋白酶(ADAMTS13)，進一步研究其在血栓止血中的作用。利用脂質體將編碼ADAMTS13全長序列的重組質粒pSecTag-ADAMTS13轉染Hela細胞，用潮霉素(Hygromycin-B)篩選得到陽性克隆細胞株，并擴大培養(yǎng)，收集上清。利用 Ni-NTA瓊脂糖柱、梯度咪唑淋洗法純化蛋白，SDS-PAGE和Western blotting鑒定純化產品純度和免疫學活性，采用GST-His雙抗夾心法測定蛋白剪切活性。結果顯示，成功獲得一株能恒定分泌重組ADAMTS13蛋白的細胞株ADAMTS2-4，每1 L培養(yǎng)上清可純化得到5.8 mg重組蛋白。Western blotting結果顯示，ADAMTS13多抗能與重組蛋白在190 kDa處顯單一條帶，并且蛋白具有6.4 U/mL的剪切活性(每毫升正常人混合血漿中ADAMTS13活性為1 U)。重組蛋白具有較好的免疫原活性和酶活性，為進一步研究ADAMTS13作用機理和運用奠定了良好的基礎。

血管性血友病因子裂解蛋白酶，真核表達，重組蛋白

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.

Keywords:von Willebrand factor cleaving protease, eukaryotic expression, recombinant protein

血管性血友病因子裂解蛋白酶(a disintegrin-like and metalloprotease with thrombospondin type 1 motif，ADAMTS13，也稱VWF-CP)是1996年發(fā)現(xiàn)的一種血漿金屬蛋白酶，它主要將血漿中大分子量的血管性血友病因子(von Willebrand factor，VWF)多聚體降解成為黏附活性低的小分子片段[1]。VWF是血漿中以多聚體形式存在的糖蛋白，在血管壁發(fā)生損傷時，對血小板的黏附與聚集起到至關重要的作用[2]。……