[Prokaryotic expression and characterization of an anti-KDR single chain antibody].
OBJECTIVE
To construct and express a single chain antibody (scFv) against human vascular endothelial growth factor (VEGF) receptor KDR and characterize its biological activity.


METHODS
The restriction enzyme sites were added to the previously cloned V(H) and V(L) genes of mAb Ycom1D3 against KDR by PCR. The anti-KDR scFv gene was constructed by the splicing overlap extensive (SOE) PCR and then inserted into fusion expression vector pAYZH. The recombinant protein was expressed in E.coli 16C9 and purified with His-tag affinity chromatography. The specificity of the purified scFv was examined by ELISA and FACS.


RESULTS
DNA sequencing indicated that the cloned scFv gene consisted of 729 bp, encoding 243 amino acids. After induction in low-phosphate medium of AP5, a new protein band with relative molecular mass (M(r)) of 30 000 appeared on gel of SDS-PAGE and on nitrocellulose membrane of Western blot, which was consistent with the theoretically predicted value. Anti-KDR scFv was expressed in the form of inclusion body, which accounted for 20% of total bacterial protein. ELISA and competitive immunofluorescence binding test showed that the anti-KDR scFv had the same binding activity as mAb Ycom1D3 and that it could block VEGF/KDR interaction effectively.


CONCLUSIONS
Recombinant anti-KDR scFv gene has been successfully constructed and expressed in E.coli 16C9, which lays the foundation for its diagnostic and therapeutic application.
Li
Rong
R
State Key Laboratory of Experimental Hematology, Institute of Hematology, CAMS & PUMC, Tianjin 300020, China.
Xiong
Dong-Sheng
DS
Xu
Yuan-Fu
YF
Shao
Xiao-Feng
XF
Fan
Dong-Mei
DM
Zhu
Zhen-Ping
ZP
Yang
Chun-Zheng
CZ
D000818:Animals; D000906:Antibodies / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D001483:Base Sequence; D001667:Binding, Competitive; D004926:Escherichia coli / Q000235:genetics; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D011387:Prokaryotic Cells / Q000276:immunology* / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D012995:Solubility; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism
D000906:Antibodies; D055767:Immobilized Proteins; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2