利用抗原结合多肽嫁接抗体技术制备抗hCG单域抗体

Preparation of anti-hCG single domain antibody by antibody grafting technique using an antigen-binding peptide

摘要:

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。

Abstract:

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.