Chen YanMin, Bai Yin, Guo XiaoChen, Wang WenFei, Zheng Qi, Wang FuXiang, Sun Dejun, Li DeShan, Ren GuiPing, Yin JieChao
College of Life Sciences, Northeast Agricultural University, Harbin 150030, People's Republic of China.
College of Life Sciences, Northeast Agricultural University, Harbin 150030, People's Republic of China; The 211th Hospital of People's Liberation Army, Harbin 150080, People's Republic of China.
Biologicals. 2016 Jul;44(4):271-275. doi: 10.1016/j.biologicals.2016.05.003. Epub 2016 May 30.
A CDR3 mutant library was constructed from a previously isolated anti-HBV neutralizing Homo sapiens scFv-31 template by random mutant primers PCR. Then the library was displayed on the inner membrane surface in Escherichia coli periplasmic space. Seven scFv clones were isolated from the mutant library through three rounds of screening by flow cytometry. Competition ELISA assay indicates that isolated scFv fragments show more efficient binding ability to HBV PreS1 compared with parental scFv-31. HBV neutralization assay indicated that two clones (scFv-3 and 59) show higher neutralizing activity by blocking the HBV infection to Chang liver cells. Our method provides a new strategy for rapid screening of mutant antibody library for affinity-enhanced scFv clones and the neutralizing scFvs obtained from this study provide a potential alternative of Hepatitis B immune globulin.
通过随机突变引物PCR，从先前分离的抗HBV中和性人源单链抗体片段（scFv）-31模板构建了一个CDR3突变文库。然后该文库展示于大肠杆菌周质空间的内膜表面。通过三轮流式细胞术筛选，从突变文库中分离出7个单链抗体片段克隆。竞争ELISA分析表明，与亲本scFv-31相比，分离得到的单链抗体片段对HBV PreS1具有更强的结合能力。HBV中和试验表明，两个克隆（scFv-3和59）通过阻断HBV对Chang肝细胞的感染表现出更高的中和活性。我们的方法为快速筛选突变抗体文库以获得亲和力增强的单链抗体片段克隆提供了一种新策略，并且本研究中获得的中和性单链抗体为乙型肝炎免疫球蛋白提供了一种潜在的替代品。
