一种噬菌体质粒的构建及其在抗体库模型筛选中的应用

Construction of a phage plasmid and application in model selection of phage antibody library

目的噬菌体展示技术(Phage display technology,PDT)是一种独立于任何免疫系统并在体外进行筛选多肽或者抗体的一种技术,可以从噬菌体抗体库中筛选得到特异性结合的单克隆抗体(monoclonal antibody)。本研究提出了一种应用于噬菌体抗体库筛选的质粒构建方法,旨在提高噬菌体展示技术的筛选效率。方法首先将编码整个抗体结合域的基因(以单链可变片段scFv的形式)融合到基因III,并在融合噬菌粒scFv-PIII间引入牛凝血酶(thrombin)酶切位点,通过SDS-PAGE及Western Blot实验验证牛凝血酶特异性洗脱效果,同时利用ELISA(酶联免疫吸附实验)及噬菌体滴度检测特异性洗脱后单链抗体的结合活性,最后根据该质粒设计一种新的模型筛选方法,验证筛选效率。结果本研究成功构建目标质粒并利用该质粒进行模型筛选,证实筛选效率得到提高。结论在构建噬菌体抗体库的噬菌粒载体上添加牛凝血酶特异性酶切位点的筛选方法,可以减少筛选轮次高效筛选出特异性噬菌体抗体。

Objective Phage display technology is independent from any immune system and selects peptides and antibodies in vitro from phage antibody library,which can obtain monoclonal antibodies specifically binding to antigen from phage antibody library.We proposed a method of constructing plasmid applying to phage selection in order to increase selection efficiency.Methods We first combined the gene coding antibody binding domain in the form of scFv and gene III,and introduced thrombin site between scFv and phage plasmid PIII.Then we determined the effect of thrombin elution by SDS-PAGE and Western Blot,and determined specific affinity between scFv and antigen by ELISA and phage titer.Finally we designed a new model selection method based on new phage palsmid to test selection effict.Results The target plasmid was successfully constructed and used for model selection,which proved that the screening efficiency was improved.Conclusions These results showed that the selection method of introducing thrombin site in phage plasmid vector that constructing phage antibody library could efficiently select specific antibody by reducing the rounds.