南方医科大学学报2011，Vol.31Issue(2)：308-312,5.
快速构建哺乳动物细胞表面展示的全长人抗体基因库
Rapid construction of a mammalian cell surface display library of full-length human antibodies
摘要
Abstract
Objective To construct a mammalian cell surface display library of full-length human antibodies. Methods The total RNA was isolated from human peripheral blood mononuclear cells (PBMCs), and the genes encoding the heavy chain variable regions and kappa light chains (VH and Ck) of the antibodies were amplified by RT-PCR. The amplified VH and Ck gene sequences were separately inserted into the vector pDGB-HC-TM. The ligation mixtures were transformed into competent E.coli DH5α cells to construct the antibody libraries, and the library sizes and diversity were analyzed. The library DNAs were transfected into CHO cells and the expression of the full-length human antibodies on the surface of CHO cells was analyzed by flow cytometry. Results The heavy chain gene library constructed showed a diversity of 2.6×105, and the kappa light chain gene library had a diversity of 2.0×105. Sequence analysis of 10 clones randomly selected from the constructed heavy chain gene library and 10 from the light chain gene library showed that 8 heavy chain clones and all 10 light chain clones contained correct open reading frames. Flow cytometry demonstrated that all the 18 clones expressed full-length antibodies, which could be detected on CHO cell surfaces. After co-transfection of the heaw chain and light chain gene libraries into CHO cells, the expression offull-length antibodies on CHO cell surfaces was detected with the positive cells reaching 31.5. Conclusion A full-length human mammalian display antibody library with a combinatory diversity of 5.2× 1010 can be constructed in two weeks, which allows the display of full-length antibodies on mammalian cell surface.关键词
快速构建/哺乳动物细胞展示/全长人源抗体库Key words
library construction/ antibody library/ mammalian cell surface display/ full-length human antibody library分类
医药卫生引用本文复制引用
贺微,李长征,陈振瑞,周烨,谭万龙,姜世勃,周志刚,刘叔文,周辰..快速构建哺乳动物细胞表面展示的全长人抗体基因库[J].南方医科大学学报,2011,31(2):308-312,5.基金项目
香港Dgen Biotech Ltd科研基金 （）
南方医科大学杰青培育基金 （）
