Antigen Specific VNAR Screening in Whitespotted Bamboo Shark (Chiloscyllium plagiosum) with Next Generation Sequencing

IgNAR exhibits significant promise in the fields of cancer and anti-virus biotherapies. Notably, the variable regions of IgNAR (VNAR) possess comparable antigen binding affinity with much smaller molecular weight (~12 kDa) compared to IgNAR. Antigen specific VNAR screening is a changeling work, which limits its application in medicine and therapy fields. Though phage display is a powerful tool for VNAR screening, it has a lot of drawbacks, such as small library coverage, low expression levels, unstable target protein, complicating and time-consuming procedures. Here we report VNAR screening with next generation sequencing (NGS) could effectively overcome the limitations of phage display, and we successfully identified approximately 3000 BAFF-specific VNARs in Chiloscyllium plagiosum vaccinated with the BAFF antigen. The results of modelling and molecular dynamics simulation and ELISA assay demonstrated that one out of the top five abundant specific VNARs exhibited higher binding affinity to the BAFF antigen than those obtained through phage display screening. Our data indicates NGS would be an alternative way for VNAR screening with plenty of advantages. Overall design: Next Generation Sequencing results of VNAR in immune BAFF antigen and untreated Whitespotted Bamboo Shark (Chiloscyllium plagiosum) Different primer pairs were used for VNAR libraries' preparations. NR means the library was prepared with primers IgNAR_NF&IgNAR_NR. OR means the library was prepared with primers IgNAR_NF&IgNAR_OR.

IgNAR在癌症与抗病毒生物治疗领域展现出显著应用潜力。值得注意的是，IgNAR的可变区（VNAR）的抗原结合亲和力与完整IgNAR相当，但其分子量仅约12 kDa，远小于完整IgNAR。
抗原特异性VNAR筛选是一项极具挑战性的工作，这限制了其在医药与治疗领域的应用。尽管噬菌体展示技术是VNAR筛选的有力工具，但该技术存在诸多缺陷：文库覆盖度低、表达水平不佳、靶蛋白不稳定、操作流程复杂且耗时。
本研究证实，采用下一代测序（Next Generation Sequencing, NGS）进行VNAR筛选可有效克服噬菌体展示技术的局限，并在经BAFF抗原免疫的白点竹鲨（Chiloscyllium plagiosum）中成功筛选得到约3000株BAFF特异性VNAR。
建模、分子动力学模拟及酶联免疫吸附试验（ELISA）结果显示，丰度排名前五的特异性VNAR中，有一株与BAFF抗原的结合亲和力显著高于噬菌体展示技术筛选得到的VNAR。
本研究数据表明，NGS可作为VNAR筛选的替代方案，具备诸多优势。
整体实验设计：经BAFF抗原免疫与未处理的白点竹鲨（Chiloscyllium plagiosum）体内VNAR的下一代测序结果。
实验采用不同引物对构建VNAR文库：NR组代表以引物IgNAR_NF与IgNAR_NR制备的文库；OR组代表以引物IgNAR_NF与IgNAR_OR制备的文库。