A novel strategy for rapid construction of libraries of full-length antibodies highly expressed on mammalian cell surfaces

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 3 10 10 .

Identification of HBsAg-specific antibodies from a mammalian cell displayed full-length human antibody library of healthy immunized donor

“…Vector pDGB-HC-TM and vector pDGB4 had already been constructed as described previously. 19,22 Miniprep, maxiprep and gel extraction Using the kits bought from Axygen (Union City, NJ, USA), experiments were performed according to the manufacturer's instructions.…”

“…In a 50 ml tube, 200 nM forward and reverse primers were mixed with 2 ml of RT products and 25 ml of 23 Master Mix. 19 Amplification conditions were as follows: 94 uC for 5 min to denature the template, followed by 35 cycles of 30 s at 94 uC, 30 s at 55 uC and extension at 72 uC for 1 min per 1 kb length of DNA to be amplified, ending with 7 min of extension at 72 uC. PCR products were separated by electrophoresis on a 1% agarose gel and purified.…”

“…19 Using total RNA isolated from donor's PBMC as template, the full-length kappa LC repertoire and variable domain of HC (VH) repertoire were amplified by two-step RT-PCR procedure. PCR primers were designed according to the sequence information of V-base as described (http://vbase.mrccpe.cam.ac.uk/) and contained comparable restriction endonucleaserecognizing sequences at the 59-ends.…”

“…PCR primers were designed according to the sequence information of V-base as described (http://vbase.mrccpe.cam.ac.uk/) and contained comparable restriction endonucleaserecognizing sequences at the 59-ends. 19,20 The forward primers, from 59-to 39-, contain restriction endonuclease-recognizing sequences, Kozak sequence (CCACC), start codon and gene-specific sequences. The reverse primers contain restriction endonuclease-recognizing sequences and gene-specific sequence.…”

“…15 Using the vector pDGB-HC-TM and the enzymes BsmBI and SfiI, we have also developed a method to construct large full-length antibody libraries with sizes of 10 10 -10 11 within 2-8 weeks. [17][18][19][20] Using the one-step fourway ligation method and the vector pDGB4, we can construct highly efficient antibody libraries with sufficient stability to display fulllength antibodies on mammalian cell surfaces. When coupled with the Flp recombinase-mediated integration (Flp-In) 21 system, each cell displays only one kind of antibody molecule, 15,22,23 which may be helpful for the screening of high-affinity antibodies with biological function.…”