Luo Ying, Li Yanping, He Qinghua, Tu Zhui
State Key Laboratory of Food Science and Resources, Nanchang University, Nanchang 330047, Jiangxi, China.
College of Food Science and Technology, Nanchang University, Nanchang 330047, Jiangxi, China.
Sheng Wu Gong Cheng Xue Bao. 2025 Apr 25;41(4):1500-1514. doi: 10.13345/j.cjb.240940.
This study is designed to address the development, synthesis, and screening of non-animal-derived nanoantibody libraries. Furthermore, it seeks to elucidate the impact of framework region selection and complementarity-determining region (CDR) design on the characteristics of synthesized nanoantibody libraries. These investigations aim to establish a robust theoretical and technical foundation for enhancing the efficacy, diversity, and practical applicability of synthetic nanoantibody libraries. In this study, a new framework (IGHV3S6501-IGHJ401) was identified based on the high-throughput sequencing results of natural nanobodies, and degenerate primers were designed based on the frequency of amino acids at each position in the complementarity-determining region (CDR) region to synthesize the coding fragments of nanobodies by overlap PCR. After 40 times of electro-transformation, a single-frame synthesized nanobody library (SS-Library) containing 6×10 clones was obtained, and the titer of the library was demonstrated to be 10 PFU/mL after rescue by the helper phage M13K07. Random 48 single colonies were picked for PCR, which revealed an insertion rate of 95.8%. Sanger sequencing results showed that 38 clones had complete sequences, none of which showed cysteines or stop codons, and no identical sequences appeared, suggesting that the library had higher diversity. The library was screened and validated with three antigens, including bovine serum albumin (BSA), acetylcholinesterase (AchE), and immunoglobulin G (IgG). Finally, 2 nanobodies against BSA, 10 against AchE, and 15 against IgG were obtained. One positive clone of each antigen was singled out for recombinant expression, and the results showed that all the three nanobodies were expressed in a soluble form. The binding activity of recombinantly expressed nanobodies was evaluated using indirect enzyme-linked immunosorbent assay (ELISA) and bio-layer interferometry (BLI). The results demonstrated that the anti-AChE and anti-IgG nanobodies exhibited specific binding to their respective antigens, with affinity constants (KD) of 294 nmol/L and 250 nmol/L, respectively. The nanobody synthetic library preparation method proposed in this study is simple and easy to use with low preference, and it is expected to be a universal nanobody discovery platform for the preparation and development of lead specific nanobodies.
本研究旨在解决非动物源纳米抗体文库的开发、合成及筛选问题。此外，该研究试图阐明框架区选择和互补决定区（CDR）设计对合成纳米抗体文库特性的影响。这些研究旨在为提高合成纳米抗体文库的功效、多样性及实际适用性建立坚实的理论和技术基础。在本研究中，基于天然纳米抗体的高通量测序结果鉴定出一个新的框架（IGHV3S6501 - IGHJ401），并根据互补决定区（CDR）区域各位置氨基酸的频率设计简并引物，通过重叠PCR合成纳米抗体的编码片段。经过40次电转化后，获得了一个包含6×10个克隆的单框架合成纳米抗体文库（SS - Library），经辅助噬菌体M13K07拯救后，文库滴度显示为10 PFU/mL。随机挑选48个单菌落进行PCR，结果显示插入率为95.8%。桑格测序结果表明，38个克隆具有完整序列，其中均未出现半胱氨酸或终止密码子，也未出现相同序列，这表明该文库具有较高的多样性。使用三种抗原对该文库进行筛选和验证，这三种抗原包括牛血清白蛋白（BSA）、乙酰胆碱酯酶（AchE）和免疫球蛋白G（IgG）。最终，获得了2个抗BSA纳米抗体、10个抗AchE纳米抗体和15个抗IgG纳米抗体。每种抗原挑选出一个阳性克隆进行重组表达，结果表明所有这三种纳米抗体均以可溶形式表达。使用间接酶联免疫吸附测定（ELISA）和生物层干涉术（BLI）评估重组表达纳米抗体的结合活性... 显示全部
结果表明，抗AchE和抗IgG纳米抗体分别与其各自的抗原表现出特异性结合，亲和常数（KD）分别为294 nmol/L和250 nmol/L。本研究提出的纳米抗体合成文库制备方法简单易用，偏好性低，有望成为用于制备和开发先导特异性纳米抗体的通用纳米抗体发现平台。
