The advances in high-throughput DNA sequencing and recombinant antibody technologies has presented new methods for characterizing antibody repertoires and significantly increased our understanding on the functional role of antibodies in immunity and their use in diagnostics, vaccine antigen design and as biological therapeutics. A subset of Bos taurus antibodies possesses unique ultra-long third complementary-determining region of the heavy chain (CDRH3) and are of special interest because they are thought to have unique functional abilities of broadly neutralizing properties – a functional role that has not been fully explored in vaccine development. Next generation sequencing technologies that are widely used to profile immunoglobulin (Ig) repertoires are based on short-read methods such as the Illumina technology. Although this technology has worked well in sequencing Ig V-D-J regions of most jawed vertebrates, it has faced serious technical challenges with sequencing regions in bovine Ig bearing ultra-long CDRH3 sequences, which are longer than 120 bp. To overcome this limitation, we have developed a sequencing strategy based on nested PCR products that allows sequence assembly of full-length bovine Ig heavy-chain (IgH) V-D-J regions. We have used this strategy to sequence IgH V-D-J regions of two Bos indicus breeds, Ankole and Boran. We confirm the presence of ultra-long CDRH3 sequences in IgG transcripts in both African cattle breeds, and provide preliminary evidence for differences and preferences in germline V_H, D_H and J_H allele gene usage as well as differences in the length of the VH region in the two bovine breeds. Our method provides tools that should allow more robust analyses of ultra-long CDRH3 sequences aiding antibody and epitope discovery in different cattle breeds and their role in mediating immunity.

高通量 DNA 测序和重组抗体技术的进步提供了表征抗体库的新方法，并显着增加了我们对抗体在免疫中的功能作用及其在诊断、疫苗抗原设计和生物治疗中的应用的理解。Bos 金牛座的一个子集抗体拥有独特的超长重链第三互补决定区 (CDRH3)，并且具有特殊的意义，因为它们被认为具有广泛中和特性的独特功能能力——这一功能作用尚未在疫苗开发中得到充分探索。广泛用于分析免疫球蛋白 (Ig) 库的新一代测序技术基于短读长方法，例如 Illumina 技术。尽管这项技术在对大多数有颌脊椎动物的 Ig VDJ 区域进行测序方面效果很好，但它在对牛 Ig 中带有超长 CDRH3 序列（长度超过 120 bp）的区域进行测序方面面临着严峻的技术挑战。为了克服这个限制，我们开发了一种基于嵌套 PCR 产品的测序策略，允许对全长牛 Ig 重链 (IgH) VDJ 区域进行序列组装。我们已经使用这种策略对两个 IgH VDJ 区域进行了测序Bos indicus品种，Ankole 和 Boran。我们确认了两个非洲牛品种的 IgG 转录本中存在超长 CDRH3 序列，并为种系 V _H、D _H和 J _H等位基因基因使用的差异和偏好以及 VH 长度的差异提供了初步证据两个牛品种的地区。我们的方法提供的工具应该允许对超长 CDRH3 序列进行更可靠的分析，帮助不同牛品种中的抗体和表位发现及其在介导免疫中的作用。