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

作　　者：陈奇[1] 万翠香[1,2] 涂追[1] 谭强来[1] 熊勇华[1,2] 陶勇[2] 

机构地区：[1]南昌大学食品科学与技术国家重点实验室,江西南昌330047 [2]南昌大学中德联合研究院,江西南昌330047

出　　处：《微生物学通报》2014年第9期1816-1821,共6页Microbiology China

基　　金：国家自然科学基金项目(No.31271863);江西省自然科学基金项目(No.20122BAB214010);江西省教育厅青年科学基金项目(No.GJJ12142)

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。[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 4rounds 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-Listeriamonocytogenes specific phage display antibodies under the optimized conditions in this study.Subtractive panning was efficient and necessary to eliminate non-specific clones.

关 键 词：噬菌体抗体库 单增李斯特菌 消减筛选法 单域重链抗体 

分 类 号：TS201.3[轻工技术与工程—食品科学]