单框架全合成纳米抗体文库的构建与鉴定

Construction and characterization of single-framework fully synthetic nanobody libraries

摘要:

本研究旨在提供一种非动物源纳米抗体文库设计、合成及筛选方案，探索框架区的选择及互补决定区(complementarity determining region,CDR)的设计对于合成纳米抗体文库的影响，为增加合成纳米抗体文库的有效性、多样性及实用性奠定理论和技术基础。本研究基于天然纳米抗体的高通量测序结果，确定了1个新的框架(IGHV3S65*01-IGHJ4*01)，依据CDR中每个位置氨基酸的出现频率，分别设计简并引物，通过重叠延伸PCR (overlap PCR)合成纳米抗体编码片段。经过40次电转化，获得了包含6×10⁹个克隆的全合成纳米抗体文库(single-frame synthesized nanobody library,SS-Library)，经辅助噬菌体M13K07救援后，展示文库滴度为10¹³ PFU/mL。随机挑取48个单菌落进行菌落PCR验证，插入率达95.8%，Sanger测序结果显示38个克隆序列完整，均未见半胱氨酸以及终止密码子，且未出现完全相同的序列，提示文库多样性良好。以牛血清白蛋白(bovine serum albumin,BSA)、乙酰胆碱酯酶(acetylcholinesterase,AchE)、小鼠IgG (immunoglobulin G,IgG)为靶标，对文库进行筛选验证，分别获得了2种针对BSA、10种针对AchE以及15种针对IgG的纳米抗体。每种抗原分别挑取了1个阳性克隆进行重组表达，结果表明3种纳米抗体均为可溶性表达。采用间接酶联免疫吸附实验(enzyme-linked immunosorbent assay,ELISA)和生物膜干涉技术(bio-layer interferometry,BLI)对重组表达纳米抗体结合活性进行了测定，结果表明，抗AchE和IgG的纳米抗体可以特异性结合相应抗原，亲和力常数分别为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行、偏好性低，有望成为一种通用的纳米抗体发现平台，用于先导特异性纳米抗体的制备与开发。

Abstract:

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 (IGHV3S65*01-IGHJ4*01) 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 6×10⁹ clones was obtained, and the titer of the library was demonstrated to be 10¹³ 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.