肺癌靶向血栓蛋白hu3D3V_H-tTF的表达及活性鉴定

Abstract

目的制备一种用于肺癌血管靶向栓塞治疗的融合蛋白hu3D3VH-tTF,并鉴定其生物学活性。方法利用重叠PCR技术构建tTF与hu3D3VH的融合基因,克隆至表达载体pET22 b(+),在E.coliBL21(DE3)中表达,镍亲和色谱柱纯化目的蛋白。ELISA检测融合蛋白hu3D3VH组分与肺腺癌细胞A549选择性结合活性,凝血实验和FⅩ活化实验鉴定融合蛋白tTF组分的促凝血活性。结果获得序列正确的hu3D3VH/tTF/pET22 b(+)重组子,融合蛋白在E.coliBL21(DE3)中高效表达。纯化后的融合蛋白与肺腺癌细胞A549具有选择性结合活性,并能活化FⅩ、有效促发血液凝固。结论成功构建hu3D3VH/tTF/pET22 b(+)重组子,hu3D3VH/tTF融合蛋白具有hu3D3VH的选择性结合能力同时具有TF的促凝血活性,为开展选择性肺肿瘤血管血栓性栓塞研究奠定了基础。 【英文摘要】 Purpose To prepare the fusion protein of hu3D3VH-tTF for thrombosis targeting therapy of lung cancer and to analyze its biological activities.Methods Fusion gene hu3D3VH-tTF was constructed by overlap PCR,cloned into expression vector pET22 b(+),and expressed in E.coli BL21(DE3).The fusion protein hu3D3VH-tTF was purified through Nickel-affinity chromatography column.The selective binding activity of the hu3D3VH moiety of fusion protein was analyzed using ELISA and the coagulation activity of the tTF moiety...