基于噬菌体展示天然纳米抗体库的人源白脂素纳米抗体制备研究

Preparation of Human Asprosin Nanobodies Based on a Natural Nanobody Phage Display Library

摘要:

【目的】 白脂素（asprosin，ASP）是一种由原纤维蛋白1（FBN1）裂解产生的脂肪因子，其水平异常与2型糖尿病、肥胖症等疾病密切相关，对健康影响显著，因此对ASP的监测具有重要意义。作者旨在通过噬菌体展示技术筛选特异性结合ASP的纳米抗体，为ASP的研究提供一种新的抗体元件。 【方法】 利用在线工具ABCpred和NovoPro预测ASP的抗原表位，并与牛血清白蛋白（BSA）偶联作为包被抗原ASP-Epitope-BSA。通过5轮生物亲和淘选，从噬菌体展示天然纳米抗体库中筛选特异性结合ASP的纳米抗体。在大肠杆菌中诱导表达筛选出的纳米抗体，测定其与ASP-Epitope-BSA和ASP全抗原的半数效应浓度（EC₅₀）并进行特异性分析。最后，进行同源建模和分子对接研究，分析纳米抗体与ASP的结合模式。 【结果】 作者成功筛选出1株特异性结合ASP的纳米抗体VHH-19，其在大肠杆菌中的诱导表达量为2.4 mg/dL（以LB培养基计）。性能鉴定结果显示，VHH-19与ASP-Epitope-BSA和ASP全抗原的EC₅₀分别为（87.49±0.27）、（128.94±0.48） ng/mL，且具有较好的特异性。同源建模和分子对接结果显示，ASP被吸附在由VHH-19的CDR1区、CDR3区形成的活性口袋内，形成一种夹心式结合模式，其中ASP的Try116、Gly126、Lys115、Glu124与VHH-19的Glu107、Arg31、Arg33分别形成氢键与静电相互作用。 [结论] 作者基于噬菌体展示天然纳米抗体库，通过生物亲和淘选方法快速筛选并获得了特异性结合ASP的纳米抗体VHH-19。

Abstract:

[Objective] Asprosin (ASP), an adipokine produced from fibrillin-1 (FBN1) cleavage, is closely linked to type 2 diabetes mellitus and obesity when its level is abnormal, demonstrating significant effect on human health. Therefore, monitoring the ASP level is crucial. The author aims to screen the nanobodies specifically binding to ASP via phage display technology, offering a new antibody component for the research on ASP. [Method] The antigenic epitopes of ASP were predicted by the online tools ABCpred and NovoPro, and then coupled with bovine serum albumin (BSA) to form the coating antigen (ASP-Epitope-BSA). After five rounds of bio-panning, the nanobodies specifically binding to ASP were screened from a nanobody phage display library. The selected nanobodies were induced for expression in Escherichia coli. The half median effect concentration (EC₅₀) values with ASP-Epitope-BSA and whole ASP antigen were determined, and the specificity was evaluated. Finally, homology modeling and molecular docking were performed to study the binding mode between the nanobody and ASP. [Result] One ASP-specific nanobody (VHH-19) was successfully screened out, with the induced expression level of 2.4 mg/dL in the LB culture with Escherichia coli. Performance evaluation showed that VHH-19 had EC₅₀ values of (87.49±0.27) ng/mL and (128.94±0.48) ng/mL for ASP-Epitope-BSA and whole ASP antigen, respectively, demonstrating good specificity. Homology modeling and molecular docking revealed that ASP was adsorbed in an active pocket formed by the CDR1 and CDR3 regions of VHH-19, forming a sandwich-like binding mode. The Try116, Gly126, Lys115, and Glu124 of ASP form hydrogen bonds and electrostatic interactions with Glu107, Arg31, and Arg33 of VHH-19, respectively. [Conclusion] This study rapidly obtained the ASP-specific nanobody VHH-19 by bio-affinity panning based on a natural nanobody phage display library.