A novel strategy for rapid construction of libraries of full-length antibodies highly expressed on mammalian cell surfaces.
Development of a versatile mammalian display system is essential for the selection of functional human antibodies with high affinities. Here we described a novel strategy for rapid construction of full-length antibody libraries that could be efficiently expressed on mammalian cell surfaces. The universal vector pDGB-HC-TM was constructed by inserting multiple cloning site unique sequences recognized by restriction endonucleases BsmBI, SfiI, and BstXI for the pop-in and pop-out of genes of interest. Cytomegalovirus promoter, a commonly used promoter for high expression of proteins in a variety of mammalian cells, was used to drive expression of the inserted antibody genes and a transmembrane domain from platelet-derived growth factor receptor was fused in frame to the C-terminus of heavy chain consistent region to anchor the antibody expressed on the mammalian cell surface. Using this strategy, we constructed a full-length human antibody display library. DNA sequence analysis and expression analysis indicated that the library constructed had a combinatory expressible, detectable diversity of 6.58 x 10(10).
Zhou
Ye
Y
Institute of Genetic Engineering, Southern Medical University, Guangzhou, China.
Chen
Zhen-Rui
ZR
Li
Chang-Zheng
CZ
He
Wei
W
Liu
Shuwen
S
Jiang
Shibo
S
Ma
Wen-Li
WL
Tan
Wanlong
W
Zhou
Chen
C
D000906:Antibodies / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D005434:Flow Cytometry; D015870:Gene Expression; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D000906:Antibodies