肺癌靶向血栓蛋白hu3D3V_H-tTF的表达及活性鉴定

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。

Expression and activity characterization of thrombosis targeting protein hu3D3V_H-tTF to blood vessel of lung cancer

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 of fusion protein was detected by clotting assay and FⅩ activation assay.Results The recombinant plasmid hu3D3VH-tTF /pET22 b(+)with correct sequence was obtained and expressed highly in E.coli BL21(DE3).The purified fusion protein hu3D3VH-tTF demonstrated both the selective binding activity to human lung adenocarcinoma A549 and activities of activating FⅩ and inducing blood plasma clotting.Conclusion The recombinant hu3D3VH-tTF/pET22 b(+) was successfully established.Expressed protein hu3D3VH-tTF showed both selective binding activity of hu3D3VH and activities of tTF initiating coagulation,which provided a basis for further study on the thrombosis targeting therapy in animal model of lung cancer.