生物纳米抗体多样性及抗黄曲霉毒素纳米抗体进化

Nanobodies derived from the variable region of Heavy Chain Antibodies which naturally occur in peripheral blood and mild of camels, dromedaries and llamas. Nanobodies are wildly used for basic research, drug development and diagnosis because of the unique intrinsic properties including highly solubility, thermal stability, and remarkable resistance to extreme pH. However, it is poorly understood that how the nanobody compensate the diversity lost due to the lack of light chain. According to the present progress on the nanobody diversity and our primary data, we propose that an efficient affinity maturation mechanism may involve in the process of somatic hyper mutation and selection. ..In previous work, we immunized alpaca with an artificial antigen AFB1-BSA (Aflatoxin B1 covalently coupled to bovine serum albumin). Nanobodies specifically binding to AFB1 were isolated from the phage display library which was constructed using the lymphocytes from the last blood sample. ..In this project, the deep sequencing technology will employed to analysis the diversity and abundance of nanobody repertoire, as well as the dynamic development during antigen immunization. Then, the lineage of the nanobodies that recognize a specific epitope would be identified by comparing the nanobody repertoire sequences. Several clones of the identified nanobodies will be selected, and the coding DNA sequences will be optimized on the codon usage bias before gene synthesis. The nanobodies will be expressed, purified and characterized. The deep sequencing data will be combined with the bioassay results of the recombinant nanobody to explore the pattern(s) of nanobody affinity maturation in vivo. The results might provide answers to how nanobodies compensate diversity lost and find out if there are other factors that critical for affinity maturation in vivo, which is useful for artificial re-design of nanobody and for generation superior nanobodies in application.

纳米抗体由天然缺失轻链的重链抗体可变区组成，具有分子量小、稳定性好等优点。虽然纳米抗体在基础研究、生物制药及诊断等领域已有广泛的应用，但是我们对于纳米抗体通过何种机制补偿由于缺失轻链造成的多样性降低等问题仍然缺乏了解。结合现有关于纳米抗体多样性的解释及其它物种的研究，我们推测纳米抗体的体内进化(突变和选择过程)可能存在某种更加高效的机制。.前期研究采用黄曲霉毒素B1(AFB1)人工抗原免疫羊驼，通过噬菌体展示技术筛选获得了抗AFB1纳米抗体。本研究拟采用深度测序技术，研究随着人工抗原的免疫，纳米抗体库的多样性、丰度及其变化，在此基础上，鉴别出针对AFB1的抗体家系；采用重组表达等方法，对这些特异性纳米抗体的生物活性及稳定性等特性进行表征，结合序列信息和功能表征，探索纳米抗体在体内成熟过程中的规律，研究结果可望为重链抗体多样性补偿机制提供新的解释或补充，同时为纳米抗体人工改造提供思路和方法。

纳米抗体由天然缺失轻链的重链抗体可变区组成，具有分子量小、稳定性好等优点。虽然纳米抗体在基础研究、生物制药及诊断等领域已有广泛的应用，但是我们对于纳米抗体的多样性及亲和力成熟机制和特点仍然缺乏全面的了解。. 本研究以抗黄曲霉毒素B1(AFB1)纳米抗体库为研究对象，围绕纳米抗体文库多样性，采用深度测序技术，对免疫前后纳米抗体库的多样性及结构特点进行了研究，建立了适于重链抗体可变区深度测序的方法和流程，完成了针对纳米抗体库高通量测序数据生物信息分析的计算机代码包编写，阐明了羊驼来源纳米抗体文库多样性的一些特征，主要包括胚系基因使用偏好、V(D)J重排偏好性、CDR3长度与胚系基因来源有关、氨基酸突变偏好性等，获得了大量纳米抗体序列数据，研究结果对于纳米抗体文库的多样性有了更加系统和全面的了解，可为纳米抗体及纳米抗体文库的设计和改造提供思路和启示，同时也为基于深度学习的纳米抗体设计提供了基础数据。