双峰驼重链抗体组库的基本特征研究

目的:利用二代高通量测序技术,了解双峰骆驼循环B细胞重链抗体(HCAbs)组库的组成和基本特征。方法:通过分离骆驼外周血单核细胞(PBMC),提取m RNA,利用多重PCR和Illumina Mi-seq高通量测序技术对三头双峰骆驼的重链抗体可变区进行深度测序,分析了重链抗体组库V、J基因组成、重排时末端基因删除数和V-J基因配对率,以及CDR3的长度、香农多样性指数(Shannon index)、氨基酸组成分布等基本特征。结果:鉴定出平均每头骆驼130000条有效数据和67561条独特CDR3序列,HCAbs含量较高的V基因为IGHV1S45、IGHV1S50和IGHV1S52,J基因为IGHJ4和IGHJ6,所对应的V-J基因配对含量大于40%;CDR3的长度主要分布在10-30个氨基酸之间,含量较高的氨基酸为丙氨酸、甘氨酸和半胱氨酸;CDR3区域70%以上的平均长度为20个氨基酸长度,其中V基因长度为3 bp,J基因长度分布在1-18 bp。结论:双峰骆驼B细胞重链抗体组库由巨大的、不均匀分布(以少数VJ基因克隆占大多数)的和具有高度多样性的多克隆抗体构成,较长CDR3和富含丙氨酸、甘氨酸和半胱氨酸是HCAbs的重要特征。

Fundamental Analysis of Bactrian Camel Heavy Chain Antibodies Repertoire

Objective:To understand the composition and fundamental characteristics of Bactrian Camel heavy chain antibodies repertoire through high throughput sequencing.Methods:After extracting mRNA from PBMCs, multiplex PCR and Illumina Mi-seq sequencing had been used to study the variable domain of heavy chain of heavy-chain antibodies (VHH). V and J gene usage, the gene deletion length, V-J pairing preferences, CDR3 length distribution, Shannon index, amino acid usage have been intensively analyzed.Results:In average 130000 of useful reads were captured in our Ig-seq runs and 67561 of unique complementarity-determining region 3 (CDR3) gene sequences have been identified. The most frequently used V genes in Bactrian camel heavy chain antibodies are IGHV1S45, IGHV1S50 and IGHV1S52, and that of J genes are IGHJ4 and IGHJ6. More than 40%V-J pairing events were identified in VHH. The length of CDR3 could be 10 to 30 amino acid, and in which Alanine, Glycine and Cysteine are the most abundant amino acids. More than 70 % of CDR3 are 20 amino acids long in average, among which the length of V gene is 3 bp in average, while the length of J gene is varied from1 to 18 bp.Conclusion:The antibody repertoire from Bactrian camel B-cell is composed by huge number of highly diverse polyclonal heavy chain antibodies which are dominated by a few clones with specific V and J genes. That a longer CDR3 fragment and rich in the Alanine, Glycine as well as Cysteine is the typical feature of Bactrian camel heavy chain antibodies.