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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。
Objective To clone the gene encoding human tissue factor pathway inhibitor-2 (hTFPI-2) and express it in Escherichia coli so as to obtain the active protein of interest. Methods The gene encoding human tissue factor pathway inhibitor-2 was obtained from human placenta total RNA by RT-PCR. The gene fragment was cloned into prokaryotic expression vector pET19b and transformed into host BL21. The recombinant plasmid was induced by isopropyl-β-D - Thiogalactoside (IPTG) induces the expression of the target protein. The obtained inclusion body is purified by ion exchange and renatured in vitro. The inhibitory effect of hTFPI-2 on plasmin and trypsin was determined by chromogenic substrate method. The inhibitory effect of hTFPI-2 on matrix metalloproteinase (MMP) was determined by gelatin zymography. Effect of fibrosarcoma cell invasiveness. Results The gene encoding hTFPI-2 with the correct sequence was successfully obtained. HTFP1-2 was highly expressed in E. coli as a inclusion body, accounting for 20% -30% of the total bacterial protein. After purification, refolding can be more than 90% purity of the target protein. Refolding hTFPI-2 inhibited the activity of trypsin, plasmin, MMP-2 and MMP-9, and also inhibited the invasion and metastasis of fibrosarcoma cell line HT-1080. Conclusion The prokaryotic expression system has been successfully used to obtain Active hTFPI-2.