[Construction of a phage antibody library and screening of anti-epidermal growth factor receptor variant III single chain antibody].
OBJECTIVE
To obtain specific anti-epidermal growth factor receptor variant III (EGFRvIII) single chain antibody (ScFv) by phage antibody library display system.


METHODS
The total RNA was extracted from the spleen B cells of BALB/c mice immunized with pep-3-OVA protein, and the first-strand cDNA was synthesized by reverse transcription. Antibody VH and VL gene fragments were amplified and joined to a ScFv gene with the linker. The ScFv gene was ligated into the phagemid vector pCANTAB5E, which was transformed into competent E. coli TG1. The transformed cells were then infected with M13KO7 helper phage to yield the recombinant phage to construct the phage ScFv library. Pep-3-BSA protein was used to screen the phage antibody library and ELISA carried out to characterize the activity of the antibody.


RESULTS
The VH and VL gene fragments of the antibody were about 350 bp and 320 bp in length as analyzed by agarose gel electrophoresis. The ScFv gene was 780 bp, consistent with the expected length. The recombinant phagemid with ScFv gene insert was rescued, and an immune phage ScFv library with the content of 5.0x10(6) was constructed. The recombinant ScFv phage had a titer of 3.0x10(4) cfu/ml, and the fourth phage harvest yielded 56 times as much as that of the first one. SDS-PAGE demonstrated a molecular mass of the soluble ScFv of about 28 kD. ELISA results indicated good specificity of the ScFv to bind EGFRvIII.


CONCLUSIONS
An immune phage ScFv library is successfully constructed, and the ScFv antibody fragment is capable of specific binding to EGFRvIII.
Han
Dong-gang
DG
Department of Medical Ultrasound Study, Second Affiliated Hospital, Xi'an Jiaotong University College of Medicine, Xi'an 710004, China. eastg2006@126.com
Duan
Xiao-yi
XY
Guo
You-min
YM
Zhou
Qi
Q
Wang
Quan-ying
QY
Yang
Guang-xiao
GX
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D066246:ErbB Receptors / Q000235:genetics* / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000235:genetics / Q000276:immunology; D019151:Peptide Library; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000276:immunology
D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D050505:Mutant Proteins; D019151:Peptide Library; D057127:Single-Chain Antibodies; C055966:immunoglobulin Fv; C541485:EGFR protein, mouse; D066246:ErbB Receptors