1. A novel strategy for rapid construction of libraries of full-length antibodies highly expressed on mammalian cell surfaces
- Author
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Wanlong Tan, Shibo Jiang, Ye Zhou, Shuwen Liu, Zhen-rui Chen, Chen Zhou, Chang-zheng Li, Wen-li Ma, and Wei He
- Subjects
Sequence analysis ,Genetic Vectors ,Biophysics ,Gene Expression ,Computational biology ,Transfection ,Biochemistry ,Antibodies ,Growth factor receptor ,Multiple cloning site ,Humans ,Vector (molecular biology) ,Cloning, Molecular ,Gene ,Gene Library ,Genetics ,biology ,Reverse Transcriptase Polymerase Chain Reaction ,General Medicine ,Flow Cytometry ,Restriction enzyme ,Transmembrane domain ,HEK293 Cells ,Leukocytes, Mononuclear ,biology.protein ,Antibody - Abstract
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).
- Published
- 2010
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