全人源单链抗体库噬菌体展示技术的建立  

Construction of phage display technology for complete human single-chain antibody fragment library

作　　者：张雨超[1] 丁龙飞[2] 陈健[2] 张晓燕[2] 徐建青[1] ZHANG Yu-chao;DING Long-fei;CHEN JIAN;ZHANG Xiao-yan;XU Jian-qing(School of Laboratory Medicine and Life Sciences,Wenzhou Medical University,Wenzhou 325035,Zhejiang Province,China)

机构地区：[1]温州医科大学检验医学院生命科学学院,浙江温州325035 [2]上海市公共卫生临床中心,上海201508

出　　处：《中国生物制品学杂志》2019年第1期80-84,90,共6页Chinese Journal of Biologicals

基　　金：上海市高等级生物安全病原微生物检测专业技术服务平台项目(18DZ2293000)

摘　　要：目的建立全人源单链抗体(single-chain antibody fragment,scFv)库的噬菌体展示技术。方法采集100名健康老年志愿者的外周血,5 mL/人,混合所有全血并分离外周血单个核细胞(peripheral blood mononuclear cells,PBMCs),抽提RNA,反转录合成cDNA,以其为模板,IgG类抗体基因为引物,PCR扩增获得重链可变区片段(V_H)和轻链可变区片段(V_L),然后以V_H和V_L为模板,经重叠PCR获得scFv,经sfiⅠ酶切,插入pComb3xss载体,电转后获得一定容量的抗体库;加入辅助噬菌体VCSM13过夜培养,上清液经PEG-NaCl沉淀,无菌PBS重悬噬菌体沉淀,即完成噬菌体抗体库的构建;采用Phage ELISA法进行噬菌体单克隆抗体的特异性鉴定。结果成功构建了库容为2. 6×10~9的噬菌体抗体库;随机挑选的100个噬菌体单克隆中共筛选到2个疑似H1N1株HA抗原的噬菌体抗体。结论成功构建了噬菌体抗体库的技术平台,可用于各类功能抗体的筛选。Objective To construct the phage display technology for complete human single-chain fragment(scFv)library.Methods Peripheral blood samples were collected from 100 healthy elderly volunteers,5 mL for each,and pooled,from which peripheral blood mononuclear cells(PBMCs)were separated and subjected to RNA extraction followed by cDNA synthesis.The heavy chain variable region(VH)and light chain variable region(VL)were amplified by PCR using the obtained cDNA as a template and the special sequence of IgG antibody as primer,and used as templates to obtain scFv through overlapping PCR.The constructed scFv was digested with sfi Ⅰand inserted into pComb3 xss vector,based on which a primary antibody library was constructed by electrotransformation and co-cultured with auxiliary phage VCSM13 overnight.The supernatant was precipitated with PEG-sodium chloride, and then the bacteriophage was resuspended in sterile PBS to complete the construction of phage antibody library.Phage ELISA was used to characterize the specificity of monoclonal antibody against phage.Results The human scFv library with capacity of 2.6×10^9 was constructed.Two suspected phage antibodies against HA antigen of H1N1 strain were screened from 100 randomly selected phage monoclonal clones.Conclusion The technology platform of phage antibody library is successfully constructed,which may be used for screening various functional antibodies.

关 键 词：抗体库 单链抗体 噬菌体展示 

分 类 号：R392-33[医药卫生—免疫学] Q789[医药卫生—基础医学]