基于细胞的抗单增李斯特菌单域重链抗体的筛选及鉴定

Cell-based panning and characterization of single-domain heavy chain antibody for Listeria monocytogenes

摘要:

【目的】获得针对单增李斯特菌的特异性单域重链抗体，并对筛选过程中特异性克隆的富集规律进行分析，为筛选具有种属特异性的噬菌体展示抗体提供参考。【方法】采用固相筛选技术，以热灭活的单增李斯特菌菌体为抗原，通过四轮常规筛选和一轮消减筛选，从驼源天然噬菌体展示文库中筛选针对单增李斯特菌的单域重链抗体。采用Phage-ELISA法，对后四轮筛选洗脱物中随机挑选的噬菌体进行鉴定，阳性克隆进行基因测序及结合特异性分析。通过多序列比对分析将获得的基因序列进行分组和统计。【结果】成功筛选到2株单增李斯特菌特异性的单域重链抗体。【结论】在优化的筛选条件下，基于全细胞的筛选方法能够获得特异性识别单增李斯特菌的单域重链抗体，消减筛选对于去除非特异性克隆是有效的和必要的。

Abstract:

[Objective] To obtain anti-Listeria monocytogenes specific single-domain heavy chain antibody by subtractive panning, and to analyse the enrichment rules of specific phage clones in biopanning against composition diversity and complex antigens. [Methods] The whole cell of heat inactivated Listeria monocytogenes was used as antigen to screen specific single-domain heavy chain antibodies from an alpaca non-immune phage displayed library by solid panning technique. After 4 rounds of conventional panning and 1 round of subtractive panning, a total number of 384 phage clones randomly picked from each round were characterized by Phage-ELISA, the positive clones were sequenced and the binding specificity was analyzed by Phage-ELISA. [Results] Two anti-Listeria monocytogenes specific single-domain heavy chain antibodies were obtained. [Conclusion] Whole cell-based panning strategy was feasible for isolation anti-Listeria monocytogenes specific phage display antibodies under the optimized conditions in this study. Subtractive panning was efficient and necessary to eliminate non-specific clones.