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| Construction and Screening of Phage Antibody Libraries Against Epidermal Growth Factor Receptor Variant Type III |
| ZHOU Min,SHI Bi-zhi,GU Jian-ren,LI Zong-hai |
| State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Medical School of Shanghai Jiaotong University, Shanghai 200032, China |
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Abstract Objective: To construct a phage display library of single chain variable fragment (scFv) antibodies and to screen a scFv antibody against epidermal growth factor receptor variant type III (EGFRvIII). Method: Balb/c mice were immunized by recombinant EGFRvIIIex and NIH3T3/EGFRvIIIex cell line. Genes of variable heavy (VH) and variable light (VL) chains of antibodies were prepared by RT-PCR from the splenic cells of immunized mice and further artificially joined with a flexible peptide linker, then ligated into the phagemid vector pCANTAB 5E. The phagemides containing scFv were transformed into electrocompetent Hpd3cells. The recombinant phages were enriched through 3 rounds of affinity panning and the anti-EGFRvIII phage scFv clones were identified by ELISA. The fourth round of panning performed at a higher stringency of selection, which used a capture concentration of 0.5μg or 0.05μg. After identified by ELISA, one positive clone was sequenced and transfected into E.coli HB2151 cells to express. Result: A phage display library with a complexity of approximately 7.9×107 was constructed. After the fourth round of panning performed at a increasing stringency of selection, higher affinity clones were obtained. Sequencing analysis showed that the anti-EGFRvIII scFv was 807 base pairs coding 268 amino acids. The soluble scFv exhibited the binding activity to purified recombinant EGFRvIIIex protein and EGFRvIII overexpression cell line. Conclusion: The successful preparation of anti-EGFRvIII scFv will provide an EGFRvIII targeted molecule for the development of antibody-based drugs.
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Received: 22 December 2009
Published: 29 April 2010
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