By using an RAD peptide display system derived from the ATPase domain of recombinase RadA of Pyrococcus furiosus, an anti-hCG antibody-like molecule was prepared by grafting an hCG-binding peptide to the RAD scaffold. After linking to sfGFP gene, a gene of hCG peptide-grafted RAD was synthesized and cloned into a bacterial expression vector (pET30a-RAD/hCGBP-sfGFP). The vector was transformed into Escherichia coli, and expression of the fusion protein was induced. After isolation and purification of the fusion protein, its binding affinity and specificity to hCG were determined by using a process of immunoabsorption followed by GFP fluorescence measurement. A comparison of hCG-binding activity with a similarly grafted single-domain antibody based on a universal scaffold was performed. The measurement of hCG-binding affinity and specificity revealed that the grafted RAD has an optimally high binding affinity and specificity to hCG, which are better than the grafted single-domain antibody. Moreover, the affinity and specificity of grafted RAD molecule are comparable to those of a commercial monoclonal antibody. In addition, the hCG-binding peptide-grafted RAD molecule has a relatively high biochemical stability, making it a good substitute for antibody with potential application.
应用基于激烈火球菌Pyrococcus furiosus 重组酶RadA 的ATP 酶结构域 (RAD 骨架) 的多肽展示体系，通过嫁接人绒毛膜促性腺激素 (hCG) 结合多肽，制备抗hCG 类抗体分子。通过合成hCG 结合多肽插入RAD 多肽展示位点的类抗体基因，成功构建了pET30a-RAD/hCGBP-sfGFP 原核表达载体，在大肠杆菌中诱导蛋白表达，分离、纯化获得类抗体蛋白，通过亲和吸附-GFP 荧光检测方法测定类抗体对hCG 的结合活性，并与应用单域抗体通用骨架制备的嫁接抗体比较活性差异。结果显示，RAD 类抗体分子对hCG 分子具有较高的亲和性和特异性，显著优于单域嫁接抗体，并与商业单克隆抗体的活性相当；同时，利用RAD 多肽展示骨架制备的抗hCG 类抗体，具有较高的生化稳定性，是一种具有应用潜力的抗体替代分子。.
Keywords: RAD peptide display; antibody-like molecule; expression and purification; hCG.