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目的提高人源化鼠抗人纤维蛋白单链抗体的亲和力和特异性。方法以人源化鼠抗人纤维蛋白单链抗体为基础,通过合成部分随机化的突变引物和PCR等方法构建HCDR3和LCDR3的混合突变库,然后利用噬菌体表面呈现技术对抗体库进行筛选,并利用本实验室建立的单链抗体-碱性磷酸酶系统进行单克隆的鉴定。结果得到4株亲和力和特异性有明显改善的人源化抗体。结论构建CDR3突变库是抗体体外成熟的一个有效方法,该研究为研制低免疫原性的导向溶栓制剂奠定了基础。
Objective To improve the affinity and specificity of humanized murine anti-human fibrin scFv. Methods Based on the humanized anti-human fibrin single chain antibody, a hybrid mutant library of HCDR3 and LCDR3 was constructed by synthesizing partial random mutagenesis primers and PCR, and then the antibody library was screened by phage display technique. Monoclonal identification was performed using the single-chain antibody-alkaline phosphatase system established in our laboratory. As a result, 4 humanized antibodies with significantly improved affinity and specificity were obtained. Conclusion The construction of CDR3 mutant library is an effective method for antibody maturation in vitro. This study lays the foundation for the development of low-immunogenic targeting thrombolytic agents.