We used the antibody grafting technology to prepare anti-hCG single-domain antibodies on the basis of antigen-binding peptide to simplify the single-domain antibody preparation process and improving the biochemical stability of peptide. By using a universal single-domain antibody backbone (cAbBCII10), CDR1 or CDR3 was replaced by the hCG-binding peptide, and the grafted antibody gene sequences were synthesized and cloned into the prokaryotic expression vector pET30a(+) in fusion with a C-terminal sfGFP gene, i.e. pET30a-(His6)-cAbBCII10-CDR1/hCGBP1-sfGFP and pET30a-(His6)-cAbBCII10-CDR3/hCGBP3-sfGFP. The recombinant plasmids were transformed into E. coli BL21(DE3), and the fusion proteins were induced by IPTG. Highly soluble recombinant fusion proteins were obtained and purified by Ni-NTA affinity column. SDS-PAGE confirmed the purified protein as the target protein. The antigen-antibody binding assay showed that both the CDR1 and CDR3 grafted antibodies have hCG-binding activities. While the titers of the two grafted antibodies were similar, the binding affinity of CDR3 grafted antibody was higher than that of CDR1 grafted protein (about 2-3 times). The grafted antibodies retained the relatively high biochemical stability of the single-domain antibody backbone and were relatively thermostable and alkaline tolerant. The obtained antibodies also had a relatively high antigen-binding specificity to hCG. This study provided a reliable experimental basis for further optimization of anti-hCG single domain antibody by antibody grafting technology using antigen-binding peptide.
本研究旨在人绒毛膜促性腺激素 (hCG) 的结合多肽的基础上应用嫁接抗体技术制备抗hCG 单域抗体，简化单域抗体制备过程，提高多肽生化稳定性。利用单域抗体通用骨架 (cAbBCII10)，以hCG 结合多肽取代互补决定区CDR1 或CDR3，合成cAbBCII10 嫁接抗体全基因序列并与sfGFP 基因序列融合后，插入到带有His 标签的原核表达载体pET30a(+) 中，成功构建了pET30a-(His6)-cAbBCII10-CDR1/hCGBP1-sfGFP 与pET30a-(His6)-cAbBCII10-CDR3/hCGBP3-sfGFP 融合蛋白表达质粒。将重组质粒转化大肠杆菌BL21 (DE3)，用IPTG 诱导表达融合蛋白，得到高表达量的可溶性融合蛋白。利用Ni-NTA 亲和柱纯化得到纯蛋白，应用SDS-PAGE 鉴定纯化的蛋白为正确表达的目标蛋白。通过抗原抗体结合实验，发现hCG 结合多肽嫁接到单域抗体通用骨架的互补决定区CDR1 或CDR3 后都有抗原结合活性，具有相似的抗体滴度，且嫁接到CDR3 后的抗原结合活性比CDR1 要高(2–3 倍)。嫁接抗体基本保留了所用单域抗体框架较为稳定的生化特性，具有一定的热稳定性和较好的碱耐受性，同时，所接入的hCG 结合片段对hCG 具有较特异的结合活性，为进一步优化抗原结合多肽嫁接抗体技术制备抗hCG 单域抗体提供了可靠的实验基础。.
Keywords: CDR1; CDR3; expression and purification; fusion protein; grafted antibody; hCG; single domain antibody.