Construction and screening of phage antibody libraries against epidermal growth factor receptor and soluble expression of single chain Fv.
- Author:
Wei-Jin SHENG
1
;
Qing-Fang MIAO
;
Yong-Su ZHEN
Author Information
1. Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Antibodies, Monoclonal;
Cell Line, Tumor;
Humans;
Immunoglobulin Light Chains;
genetics;
immunology;
Male;
Mice;
Mice, Inbred BALB C;
Peptide Library;
Receptor, Epidermal Growth Factor;
genetics;
immunology;
Single-Chain Antibodies;
genetics;
immunology
- From:
Acta Pharmaceutica Sinica
2009;44(6):597-602
- CountryChina
- Language:Chinese
-
Abstract:
Recent studies have shown that epidermal growth factor receptor (EGFR) is an important target for cancer therapy. The present study prepared single chain Fv (scFv) directed against EGFR. Balb/c mice were immunized by human carcinoma A431 cells, and total RNA of the splenic cells was extracted. VH and VL gene fragments were amplified by RT-PCR and further joined into scFv gene with a linker, then scFv gene fragments were ligated into the phagemid vector pCANTAB 5E. The phagemid containing scFv were transformed into electro-competent E. coli TG1 cells. The recombinant phage antibody library was constructed through rescuing the transformed cells with help phage M13K07. The specified recombinant phages were enriched through 5 rounds of affinity panning and the anti-EGFR phage scFv clones were screened and identified with ELISA. A total of 48 clones from the library were selected randomly and 45 clones were identified positive. After infecting E. coli HB2151 cells with one positive clone, soluble recombinant antibodies about 27 kD were produced and located in the periplasm and the supernatant. The result of sequencing showed that the scFv gene was 768 bp, which encoded 256 amino acid residues. VH and VL including 3 CDRs and 4 FRs, respectively, were all homologous to mouse Ig. The soluble scFv showed the specific binding activity to purified EGFR and EGFR located in carcinoma cell membrane. The successful preparation of anti-EGFR scFv will provide an EGFR targeted molecule for the development of antibody-based drugs and biological therapy of cancer.
