全人源抗MLAA-34单链抗体的筛选和鉴定  

Selection and identification of a fully human ScFv to MLAA-34

作　　者：张扬[1] 刘海玲[1] 姚欢 张王刚[1] ZHANG Yang;LIU Hailing;YAO Huan;ZHANG Wanggang(Department of Hematology,the Second Affiliated Hospital of Xi'an Jiaotong University,Shaanxi Xi'an 710004,China)

机构地区：[1]西安交通大学第二附属医院血液科,陕西西安710004

出　　处：《现代肿瘤医学》2021年第20期3517-3521,共5页Journal of Modern Oncology

基　　金：国家自然科学基金(编号:81270596)。

摘　　要：目的:抗体药物治疗是目前癌症治疗中很有前景的一种方法,利用前期实验得到的MLAA-34纯化蛋白作为抗原,在噬菌体抗体库中进行筛选得到抗MLAA-34的单链抗体,进行测序并鉴定其亲和力。方法:包被纯化的MLAA-34抗原蛋白于96孔板上,封闭过夜,加入1×1012 cfu噬菌体抗体库,4℃结合4 h,洗脱,洗脱后的噬菌体感染大肠杆菌TG1扩增,经过3轮淘选,淘选得到的克隆再进行Phage-ELISA的鉴定,筛选得到阳性克隆。结果:纯化的MLAA-34作为抗原,经过在噬菌体抗体库中的3轮固相筛选,噬菌体的回收得到富集,洗脱的噬菌体产率显示回收率逐级增加,富集倍数约1000倍。最后一轮筛选后挑选出188个重组噬菌体克隆,His标签蛋白作为对照蛋白,应用Phage-ELISA筛选出33个阳性克隆。结论:利用得到的MLAA-34蛋白为目标抗原,对全人源噬菌体抗体库进行淘选,应用Phage-ELISA法挑选出和MLAA-34抗原结合力的阳性噬菌体,为抗MLAA-34抗体的制备奠定了基础。Objective:Antibody-based drug therapy has become one of the most successful and promising strategies for the treatment of cancer.The purified MLAA-34 was used as the antigen,and the ScFv phage clones were selected from a fully human ScFv library and sent to DNA sequencing.Methods:The purified MLAA-34 protein was used to coat 96-well plates.The wells were blocked at 4℃overnight.Phages(1×1012 cfu)were added to the wells and incubated for 4 h at 4℃.After the elution,the eluated phages were used to infect E.coli TG1.After three rounds of biopanning,individual phages were tested for antigen binding by monoclonal Phage-ELISA.The positive clones were sent for sequencing.Results:The purified MLAA-34 was used as the antigen,and the ScFv phage clones were enriched by binding to the immobilized antigen,followed by elution and repropagation three times.After three rounds of panning,the titers increased to 109,which indicated an effective enrichment.Among the 188 positive clones,the 33 clones with the highest signals using His-tag as a control antigen were MLAA-34-specific.The same sequence of clones were excluded and bioinformatics prediction results were as expected.Conclusion:The purified MLAA-34 protein was used as antigen to isolate a fully human ScFv antibody against MLAA-34 from a large human ScFv library.This work lays a foundation for the development of anti-MLAA-34 antibody drugs.

关 键 词：MLAA-34 急性单核细胞白血病 噬菌体抗体库 

分 类 号：R733.71[医药卫生—肿瘤]