Modification and identification of a vector for making a large phage antibody library.
BACKGROUND
The large phage antibody library is used to obtain high-affinity human antibody, and the Loxp/cre site-specific recombination system is a potential method for constructing a large phage antibody library. In the present study, a phage antibody library vector pDF was reconstructed to construct diabody more quickly and conveniently without injury to homologous recombination and the expression function of the vector and thus to integrate construction of the large phage antibody library with the preparation of diabodies.


METHODS
scFv was obtained by overlap polymerase chain reaction (PCR) amplification with the newly designed VL and VH extension primers. loxp511 was flanked by VL and VH and the endonuclease ACC III encoding sequences were introduced on both sides of loxp511. scFv was cloned into the vector pDF to obtain the vector pDscFv. The vector expression function was identified and the feasibility of diabody preparation was evaluated. A large phage antibody library was constructed in pDscFv. Several antigens were used to screen the antibody library and the quality of the antibody library was evaluated.


RESULTS
The phage antibody library expression vector pDscFv was successfully constructed and confirmed to express functional scFv. The large phage antibody library constructed using this vector was of high diversity. Screening of the library on 6 antigens confirmed the generation of specific antibodies to these antigens. Two antibodies were subjected to enzymatic digestion and were prepared into diabody with functional expression.


CONCLUSIONS
The reconstructed vector pDscFv retains its recombination capability and expression function and can be used to construct large phage antibody libraries. It can be used as a convenient and quick method for preparing diabodies after simple enzymatic digestion, which facilitates clinical trials and application of antibody therapy.
Zhang
Guo-min
GM
Department of Immunology, Capital Medical University, Beijing 100069, China.
Chen
Yü-ping
YP
Guan
Yuan-zhi
YZ
Wang
Yan
Y
An
Yun-qing
YQ
D001435:Bacteriophages / Q000235:genetics*; D004077:Digoxin / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000276:immunology; D019151:Peptide Library
D007128:Immunoglobulin Fragments; D019151:Peptide Library; C055966:immunoglobulin Fv; D004077:Digoxin