人源抗转铁蛋白受体单链抗体的筛选及其构建与表达

Screening, Construction and Expression of Human Anti-TfR scFv Antibody

目的:在人源单链抗体库中筛选抗人转铁蛋白受体(TfR)单链抗体(scFv)。方法:人源展示型抗体库质粒转染293T细胞,通过第一轮流式细胞分选得到比例为0.2%的阳性细胞,提取质粒并扩增,得到的质粒转染293T细胞作为下一轮筛选所需抗体库;第二轮分选时降低抗原浓度,最后得到2个候选的scFv序列。经序列分析,选择1号单链抗体基因,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单链抗体蛋白,经Forte Bio Octet进一步测定单链抗体蛋白的亲和力。结果:经过2轮筛选获得了全新的抗TfR单链抗体序列,构建并表达了该单链抗体蛋白,该单链抗体与TfR的亲和力常数为5.57×10-7。结论:从展示型人源scFv抗体库中获得了全新的抗TfR单链抗体,该单链抗体与TfR具有较好的亲和力。

Objective：To obtain anti-transferrin receptor（TfR） scFv antibody from human secretory antibody library.Methods：The human display antibody library was transfected into 293T cells,and the 0.2％ positive cells were obtained by the first round of flow cytometry.The plasmids were extracted and transformed into E.coli DH5αcells as the secondary library,and then transfected into 293T cells.In the second round,the concentration of antigen was reduced,and two alternative scFv sequenes were obtained.1 scFv was chosen to construct the expressing vector after analyzed the sequence,and transfected into 293E cells.The supernate was collected and purified,the scFv protein was isolated by the Ni-chelating affinity and its KD value was analyzed by the FortieBio Octet.Results：The new anti-TfR scFv sequences were obtained from human scFv antibody library after two rounds screening.The expressing vector was constructed and the scFv antibody protein was purified successfully.The KD value of anti-TfR scFv was 5.57×10-7.Conclusion：We have successfully obtained new anti-TfR scFv sequence from human scFv antibody library.The scFv protein has a good affinity with TfR.