凝血靶向通用效应因子tTF/SA融合蛋白的表达及活性鉴定

Abstract

制备凝血靶向通用效应因子tTF/SA融合蛋白,并鉴定其生物学活性。利用PCR技术构建tTF与链霉亲和素SA的融合基因,克隆至表达载体pET22b(+),在E.coliBL21(DE_3)中表达,镍亲和层析柱纯化tTF/SA融合蛋白。凝血实验和FⅩ活化实验鉴定融合蛋白中tTF的活性,ELISA鉴定融合蛋白中SA与生物素Biotin结合的活性。获得序列正确的tTF/SA/pET22b(+)重组子,融合基因在E.coliBL21(DE_3)中高效表达。纯化后的融合蛋白具有活化FⅩ、引起血液凝固的能力,且能与生物素结合。融合基因已成功在E.coliBL21(DE_3)中表达,tTF/SA融合蛋白具有TF和SA活性。融合蛋白tTF/SA可作为通用效应因子,与生物素化的肿瘤组织血管特异性载体联用,实现选择性诱发肿瘤组织血管栓塞的多点治疗。 【英文摘要】 To prepare a novel fusion protein (tTF/SA) as a universal effector for targeting therapy of blood coagulation and to analyze its biological activities. The fusion gene tTF/SA was constructed by PCR, then inserted into expression vector pET22 b (+),and expressed inE. coli BL21 (DE_3). The fusion protein was purified using Nickel-affinity chromatography column. The activities of tTF moiety of the fusion protein were analyzed by clotting assay and FX activation assay, and the binding activities of Streptavidin...