人源抗BLyS单链抗体的筛选及其构建与表达

Screening,construction and expression of human anti-BLyS ScFv antibody

目的:从人源单链抗体库中筛选抗B细胞刺激因子(BLy S)的单链抗体基因、构建表达载体并实现单链抗体的表达。方法:以哺乳动物细胞展示型人源Sc Fv抗体库为起始文库,通过流式细胞术进行分选,获得荧光强度最强、比例为0.1%的阳性细胞。从候选细胞中提取质粒并转化至DH5α中进行扩增,得到质粒转染293T细胞作为下一轮筛选所需的抗体库。经过依次降低抗原浓度进行了3轮分选得到2个候选的Sc Fv序列。经序列分析,选择其中一种单链抗体基因,利用基因工程技术构建分泌型表达质粒,转染293E细胞并通过镍亲和层析纯化获得B-10 Sc Fv抗体蛋白并通过Fortie Bio Octet QK进行亲和力分析。结果:经过3轮筛选获得了全新的抗BLy S Sc Fv抗体序列,成功构建并表达了anti-Bly S Sc Fv抗体蛋白,该单链抗体与的BLy S亲和常数为3.08 nmol·L-1。结论:从哺乳动物细胞展示型人源Sc Fv抗体库中成功获得了可结合BLy S的全新单链抗体基因序列,该单链抗体与BLy S具有较高的亲和力,这为后续的活性研究以及产品开发奠定了基础。

Objective： To obtain the gene of anti-BLyS ScFv from human ScFv antibody library, construct the expression vector and express the ScFv antibody. Methods： The mammalian cell display human ScFv antibody library was used to obtain the 1% positive cells with the strongest fluorescence intensity as detected by the flow cytometry. The plasmids were extracted from the positive cells and transformed in DHSct cells as the secondary library, and then transfected into 239T cells. Two alternative SeFv sequences were obtained after 3 rounds of screening under conditions of serial dilutions of antigen concentration. One ScFv was chosen to construct the expressing vector after analyzed the sequences, and transfected into 239E cells. The supernate was collected and purified, the B-10 ScFv protein was isolated by the Ni-chelating affinity and its Kd value was analyzed by the FortieBio Octet QK. Results ： The new anti-BLyS ScFv sequences was obtained from human ScFv antibody library after 3 rounds of screening. The expressing vector was constructed and the SeFv antibody protein was purified successfully. The KD value of B10-ScFv was 3.08 nmol· L^-1. Conclusion： We have successfully got new anti-BLyS ScFv sequences with high affinity from human ScFv antibody library. This work laid a a solid foundation for subsequent study and product development.