Optimizing the host bacteria to make a large naive phage antibody library in the recombination system.
- Author:
Wei SUN
1
;
Heng LIN
;
Fang HUA
;
Zhuo-Wei HU
Author Information
1. State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China.
- Publication Type:Journal Article
- MeSH:
Adult;
Escherichia coli;
genetics;
immunology;
Genetic Vectors;
Humans;
Immunoglobulin Heavy Chains;
genetics;
Immunoglobulin Light Chains;
genetics;
Immunoglobulin Variable Region;
genetics;
Infant, Newborn;
Integrases;
metabolism;
Lymphocytes;
immunology;
Peptide Library;
Recombination, Genetic;
genetics;
Single-Chain Antibodies;
genetics;
metabolism;
Transformation, Genetic
- From:
Acta Pharmaceutica Sinica
2013;48(1):66-70
- CountryChina
- Language:Chinese
-
Abstract:
To prepare large naive phage antibody library, the host bacteria with high transformation efficiency is used in the Cre-LoxP recombination system. The variable regions of immunoglobulin light and heavy genes were amplified from lymphocytes collected from adult peripheral blood and newborn cord blood. The genes were spliced to form the single-chain variable fragments (scFv) by overlap PCR, cloned into pDAN5a vector and then transformed into XL2-blue MRF' with the Hte gene. Compared with XL1-blue strain, the size of the primary library was increased by 3.9 times. The primary library infected Cre recombinase-expressing bacteria, and the genes between phagemids created many new VH/VL combinations. The library was calculated to have a diversity of 1.7 x 10(11) and validated by the selection of antibodies against six different protein antigens. This library provides the basis for further selection of antibody-based drugs. It is the first time to report that XL2-blue MRF' can be used to improve the diversity of the library in the recombination system.
