抗VSTM1-v2细胞因子人源化单链抗体文库的构建及筛选

Gene construction and screening of humanized single chain antibody library against VSTM1-v2 cytokine

本文将鼠源VH-CDR3库(library of murine VH-CDR3)移植到特定的人源scFv(single-chain antibody fragment)VH3-VΚ1框架构建人源化scFv文库,快速筛选抗VSTM1-v2的人源化单链抗体。以抗VSTM1-v2鼠源cDNA为模板,扩增出鼠源VH-CDR3库,随后将鼠源VH-CDR3库移植到人源scFv(VH3-VΚ1)上并通过核糖体展示富集抗VSTM1-v2人源化scFv文库,该文库序列通过与原核基因表达调控等成分(N端添加T7启动子、SD序列和PelB序列、C端添加his标签序列和T7 terminal序列)拼接,构建表达型TA载体转化大肠杆菌BL21(DE3)进行可溶性表达。最终构建了一个库容达1012的人源化单链抗体文库,完成了1 000个克隆的初步鉴定,并筛选出EC50达21.35 nmol·L 1的抗VSTM1-v2人源化scFv。该研究结果,一方面获得了具有潜在应用价值的抗-VSTM1-v2先导抗体;另一方面,为快速获得人源化抗体提供了一个新技术途径。

To rapidly select potent anti-VSTMl-v2 scFv （single-chain antibody fragment） by construction and screening of a humanized scFv library in which a murine VH-CDR3 library was grafted onto a human scFv framework. A murine VH-CDR3 library was amplified from anti-VSTMl-v2 murine cDNA and grafted on human scFv （VH3-VK1） framework. Anti-VSTMI-v2 scFv templates were selected and enriched through ribosome display, TA-cloned into expression vector, and transformed into BL21 （DE3） for soluble expression of target scFv. A total of 1 000 clones were randomly picked. Positive ones were first identified using colony PCR, indirect ELISA, Western blotting and then verified with sequencing and dose response ELISA. At last an anti-VSTMl-v2 humanized scFv with good binding affinity （EC s0 = 21.35 nmol＇L-1） was selected from the humanized library of 1012 members generated in this study. This scFv antibody might have potential applications. This study provides a new approach for rapid screeninz of humanized antibodies.