凝血靶向通用效应因子tTF/SA融合蛋白的表达及活性鉴定

Expression and Characterization of Fusion Protein tTF/SA as a Universal Effector for Targeting Blood Coagulation

摘要:

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pET22 b（+），在E.coli BL21（DE₃）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/pET22 b（+）重组子，融合基因在E.coli BL21（DE₃）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.coli BL21（DE₃）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

Abstract:

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 in E.coli BL21 (DE₃). 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(SA) to Biotin(B) were analyzed using ELISA. Results: The recombinant plasmid tTF/SA/pET22 b(+) with the correct sequence was obtained. The fusion gene tTF/SA was expressed with high yield in E.coli BL21(DE₃). The purified fusion protein retain the abilities of activating FX, inducing blood coagulation, and binding Biotin. The fusion gene tTF/SA was successfully expressed in E.coli BL21(DE₃). The recombinant tTF/SA proteins retain the activities of TF and SA. The multitarget therapy of selectively inducing thrombosis in tumor blood vessels can be achieved by the combination of tTF/SA, as a universal effector, and biotinlated carriers directing to tumor blood vessels.