核糖体展示技术筛选抗呋喃唑酮单链抗体

Screening of anti-furazolidone single chain antibody by ribosome display technique

目的运用核糖体展示技术筛选高特异性抗呋喃唑酮(furazolidone,FZD)单链抗体,并进行初步分析。方法提取小鼠杂交瘤细胞总RNA,利用反转录PCR及重叠延伸(splicing by overlap extension,SOE)-PCR技术合成VH-linker-VL单链抗体文库。采用TNT T7 Quick for PCR DNA试剂盒对单链抗体文库进行体外翻译及筛选,经过三轮筛选富集目的基因,对目的基因进行原核表达及纯化后,初步分析其特异性及亲和力。结果获取1株特异性较高的单链抗体FZD-ScFv1,IC_(50)值为13.01 ng/mL,与呋喃它酮、呋喃妥因、氨基脲、呋喃西林及其衍生物之间的交叉反应率均小于1%。结论利用核糖体展示技术成功获得亲和力较好的单链抗体,该抗体可用于建立ELISA法,为FZD残留量的快速检测提供了新的思路。

Objective To screen high specific single chain antibody against furazolidone(FZD)by ribosome display technique and analyze it.Methods Total RNA was extracted from mouse hybridoma cells and VH-linker-VLsingle chain antibody library was synthesized by reverse transcription PCR and splicing by overlap extension(SOE)-PCR.The single chain antibody library was translated and screened by TNT T7 Quick for PCR DNA kit in vitro.After three rounds of screening and enrichment,prokaryotic expression and purification of the target gene,the specificity and affinity of the target gene were preliminarily analyzed.Results A single chain antibody FZD-ScFv1 with high specificity was obtained.The IC_(50)value was 13.01 ng/mL,and the cross-reaction rate between FZD-Sc Fv1 and furaltadone,nitrofurantoin,semicarbazide,furacilin and their derivatives was less than 1%.Conclusion Using ribosome display technique,the single chain antibody with good affinity can be successfully obtained,which can be used to establish ELISA and provide a new idea for rapid detection of FZD residues.