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目的制备抗尿激酶型纤溶酶原激活物受体(uPAR)人源化抗体并初步检测它们与抗原的亲和能力。方法通过计算机辅助设计的结果,合成新型抗uPAR抗体的轻链和重链可变区基因序列,通过重叠PCR方法,拼接成完整的轻链和重链基因并克隆入pIRES双向表达载体。瞬时转染293T细胞,收取细胞上清,rProtein A亲和层析法纯化目的抗体,并进行SDS-PAGE和免疫印迹鉴定,采用Biacore3000技术检测抗体与抗原的结合能力。结果成功构建5种表达载体S1~S5,纯化的抗体在还原SDS-PAGE中表现为相对分子质量约为25×103和55×103两条带;免疫印迹分析表明,该人源化抗体可与羊抗人IgG特异性结合。Biacore3000实验结果表明,S2、S4和S5抗体与抗原具有良好的亲和活性,且亲和活性分别为1.74×10-8,1.49×10-8和1.05×10-8mol/L。结论成功构建并表达了5种抗uPAR人源化抗体,其中S2、S4和S5具有良好的抗原结合能力。
Objective To prepare anti-urokinase-type plasminogen activator receptor (uPAR) humanized antibodies and to preliminary detect their affinity with antigens. Methods The light chain and heavy chain variable region gene sequences of novel anti-uPAR antibodies were synthesized by computer-aided design. The complete light and heavy chain genes were spliced into the pIRES bi-directional expression vector by overlapping PCR. The 293T cells were transiently transfected and the supernatant was collected. The target antibodies were purified by rProtein A affinity chromatography and identified by SDS-PAGE and Western blotting. The binding ability of the antibodies to the antigen was detected by Biacore 3000 technique. Results Five kinds of expression vectors S1 ~ S5 were successfully constructed. The purified antibodies showed two bands with molecular weights of about 25 × 103 and 55 × 103 in reducing SDS-PAGE. The results of immunoblotting showed that the humanized antibodies could bind with Sheep anti-human IgG specifically binds. Biacore3000 results showed that S2, S4 and S5 antibodies had good affinity with antigens, and their affinity activities were 1.74 × 10-8, 1.49 × 10-8 and 1.05 × 10-8 mol / L, respectively. Conclusion Five anti-uPAR humanized antibodies were successfully constructed and expressed. Among them, S2, S4 and S5 had good antigen-binding ability.