A Single-Chain Antibody Using LoxP511 as the Linker Enables Large-Content Phage Library Construction via Cre/LoxP Recombination.
To obtain natural or "me-better" antibodies (e.g., affinity-maturated antibodies), phage display libraries are widely used. However, the likelihood of obtaining satisfactory antibodies depends on the library content. Here, we used computer-aided design to model the use of the LoxP511 site as a linker between the heavy and light variable domains of an antibody for construction of a large single-chain fragment (scFv) antibody phage library by using the Cre/LoxP recombinant system. Then, we constructed two novel scFvs based on 2C4, namely, AH_scFv15 (15 amino acid [aa] linker; common [SG4]3 sequence) and AH_scFv21 (21-aa linker; LoxP511 sequence), to verify the use of the LoxP511 site as a linker. Our results indicate that LoxP511 could be used effectively for the construction of a large (e.g., 5 × 10(12)) phage display library of scFv antibodies from which it was possible to isolate an antibody with the same epitope as 2C4 but with higher affinity.
Department of Gynecology and Obstetrics, PLA General Hospital, Beijing, China.
Yan
Y
Zhang
Laboratory of Immunology, Institute of Basic Medical Sciences, Beijing, China Laboratory of Cellular and Molecular Immunology, Institute of Immunology, Henan University, Kaifeng, China.
Wei
W
Wang
Laboratory of Immunology, Institute of Basic Medical Sciences, Beijing, China.
Ming
M
Lv
Laboratory of Immunology, Institute of Basic Medical Sciences, Beijing, China.
Zhou
Z
Lin
Laboratory of Immunology, Institute of Basic Medical Sciences, Beijing, China.
Jing
J
Geng
Department of Gynecology and Obstetrics, PLA General Hospital, Beijing, China.
Yali
Y
Li
Laboratory of Immunology, Institute of Basic Medical Sciences, Beijing, China.
Beifen
B
Shen
Laboratory of Cellular and Molecular Immunology, Institute of Immunology, Henan University, Kaifeng, China.
Yuanfang
Y
Ma
Laboratory of Immunology, Institute of Basic Medical Sciences, Beijing, China.
Yan
Y
Li
Laboratory of Immunology, Institute of Basic Medical Sciences, Beijing, China bioqcx1982@hotmail.com Fengjiannan1970@qq.com.
Chunxia
C
Qiao
Laboratory of Immunology, Institute of Basic Medical Sciences, Beijing, China Laboratory of Cellular and Molecular Immunology, Institute of Immunology, Henan University, Kaifeng, China.
Jiannan
J
Feng
D001435:Bacteriophages / Q000737:chemistry*; D045744:Cell Line, Tumor; D000939:Epitopes / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D019426:Integrases / Q000235:genetics*; D018613:Microscopy, Confocal; D062105:Molecular Docking Simulation; D019151:Peptide Library; D010957:Plasmids / Q000378:metabolism; D000072417:Protein Domains; D011994:Recombinant Proteins / Q000737:chemistry; D011995:Recombination, Genetic; D057127:Single-Chain Antibodies / Q000737:chemistry*
D000939:Epitopes; D019151:Peptide Library; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C045073:Cre recombinase; D019426:Integrases
Cre/LoxP; large-content library; linker; recombinant; scFv