【摘要】 目的克隆人组织因子途径抑制物-2(human tissue fact0r pathway inhibitor-2,hTFPI-2)的编码基因,并在大肠杆菌中表达,获得有活性的目的蛋白。方法应用 RT-PCR 法从人胎盘总 RNA 中获得人组织因子途径抑制物-2的编码基因,将该基因片段克隆至原核表达载体 pET19b 中,转化表达宿主 BL21,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导目的蛋白的表达。获得的包涵体经离子交换法进行纯化,并使其在体外进行复性。采用发色底物法测定 hTFPI-2对纤溶酶、胰蛋白酶的抑制作用;明胶酶谱法测定对基质金属蛋白酶(matrix metalloproteinase,MMP)的抑制作用;采用人工基底膜法观察hTFP1-2对纤维肉瘤细胞侵袭能力的影响。结果成功获得了序列完全正确 hTFPI-2的编码基因,hTFP1-2在大肠杆菌中以包涵体的形式获得高效表达,占细菌总蛋白的20%～30%。经纯化、复性后可得到纯度大于90%的目的蛋白。复性后的 hTFPI-2具有抑制胰蛋白酶、纤溶酶、MMP-2和 MMP-9的活性,同时亦具有抑制纤维肉瘤细胞 HT-1080侵袭转移的能力结论采用原核表达系统高效地获得了具有活性的 hTFPI-2。更多还原

【Abstract】 Objective To clone the human tissue factor pathway inhibitor-2(hTFPI-2)gene and ex- press it by using prokaryotic expression system.Methods The hTFPI-2 coding region was obtained by RT- PCR from human placenta total RNA.The coding fragment was then inserted into prokaryotic expression vec- tor pET19b and expressed in E.coli BL21 by IPTG induction.The produced inclusion bodies were dissolved by denaturalizing chemicals,purified by ion exchange chromatograph,and refolded in air to form proper disul- fide bonds.Chromogenic and gelatin zymography methods were used to evaluate the inhibiting effects of hTFPI-2 on trypsin,plasmin and MMPs individually.The inhibitory activity of hTFPI-2 on fabrisarcoma was investigated by matrigel.Results The coding fragment of hTFPI-2 was cloned successfully and the protein was expressed as inclusion bodies which account for 20%-30% of total host protein.The refolded hTFPI-2 could inhibit the invasive ability of fibrisarcoma HT-1080 as well as activity of plasmin,trypsin and MMPs. Conclusions The activated hTFPI-2 is obtained by using prokaryotic expressed system effectively.更多还原