Wu Yu-Chang, Kipling David, Dunn-Walters Deborah
Randall Division of Cell and Molecular Biophysics, King's College London School of Biomedical Science, London, UK.
Department of Pathology, Cardiff University, Cardiff, UK.
Methods Mol Biol. 2015;1343:199-218. doi: 10.1007/978-1-4939-2963-4_16.
The B cell receptor (BCR) repertoire is highly diverse. Repertoire diversity is achieved centrally by somatic recombination of immunoglobulin (Ig) genes and peripherally by somatic hypermutation and Ig heavy chain class-switching. Throughout these processes, there is selection for functional gene rearrangements, selection against gene combinations resulting in self-reactive BCRs, and selection for BCRs with high affinity for exogenous antigens after challenge. Hence, investigation of BCR repertoires from different groups of B cells can provide information on stages of B cell development and shed light on the etiology of B cell pathologies. In most instances, the third complementarity determining region of the Ig heavy chain (CDR-H3) contributes the majority of amino acids to the antibody/antigen binding interface. Although CDR-H3 spectratype analysis provides information on the overall diversity of BCR repertoires, this fairly simple technique analyzes the relative quantities of CDR-H3 regions of each size, within a range of approximately 10-80 bp, without sequence detail and thus is limited in scope. High-throughput sequencing (HTS) techniques on the Roche 454 GS FLX Titanium system, however, can generate a wide coverage of Ig sequences to provide more qualitative data such as V, D, and J usage as well as detailed CDR3 sequence information. Here we present protocols in detail for CDR-H3 spectratype analysis and HTS of human BCR repertoires.
B细胞受体（BCR）库具有高度多样性。库多样性在中枢通过免疫球蛋白（Ig）基因的体细胞重组实现，在外周则通过体细胞超突变和Ig重链类别转换实现。在这些过程中，会对功能性基因重排进行选择，对导致自身反应性BCR的基因组合进行淘汰，并在受到刺激后对与外源抗原具有高亲和力的BCR进行选择。因此，研究不同B细胞群体的BCR库可以提供有关B细胞发育阶段的信息，并有助于揭示B细胞病理学的病因。在大多数情况下，Ig重链的第三个互补决定区（CDR-H3）为抗体/抗原结合界面贡献了大部分氨基酸。尽管CDR-H3谱型分析提供了有关BCR库总体多样性的信息，但这种相当简单的技术分析的是每个大小在约10 - 80 bp范围内的CDR-H3区域的相对数量，没有序列细节，因此范围有限。然而，罗氏454 GS FLX Titanium系统上的高通量测序（HTS）技术可以生成广泛的Ig序列覆盖范围，以提供更多定性数据，如V、D和J的使用情况以及详细的CDR3序列信息。在此，我们详细介绍人类BCR库的CDR-H3谱型分析和HTS的实验方案。
