新型抗uPAR人源化抗体的表达和活性检测

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。

Expression and activity detection of the humanized anti-uPAR antibody

Objective To prepare the humanized monoclonal antibodies against urokinase-type plasminogen activator receptor(uPAR),and preliminarily detect their affinity to uPAR.Methods L and VH genes of humanized monoclonal antibodies against uPAR were designed by the computer and synthesized by overlap PCR.Genes encoding L and H chains were connected and then cloned into vector pIRES,a bicistronic expression vector.The recombinant plasmids were transfected into 293T cells,purified antibodies through rProteinA affinity...