[Construction and expression of single-chain Fv antibody against human VEGF].
OBJECTIVE
Binding specificity of a monoclonal antibody (MAb) (gamma. k 2b) E11 which can react with the peptide of human VEGF was determined by ELISA and immunohistochemistry. The E11 primarily proved to be potential activity of suppressing tumor growth in vivo. The construction and expression of engineering antibody (ScFv) could widen application of this MAb in both diagnosis and treatment of human cancer.


METHODS
From the hybridoma cell line secreting MAb against VEGF, total RNA was prepared and used as a template for cDNA synthesis and cloning. Reverse transcription and polymerase chain reaction (RT-PCR) were used to clone the immunoglobulin variable region gene (VH, VL). The cloned VH and VL were linked by well documented flexible peptide bridging sequence (GGGGS) 3 in the VH-linker-VL orientation. The constructed ScFv gene was inserted into pET-15b and expressed in E.coli BL21 (DE3). Biding activity of ScFv was evaluated by ELISA and immunohistochemical methods after its denature and renature treatment.


RESULTS
The sequence analysis revealed that the full length of VH was 369 bp, and VL was 333 bp. According to Kabat classification, VH and VL are the member of mouse Ig variable gene heavy chain subgroup II (A) and light chain subgroup III, respectively. The ScFv was highly expressed in the form of inclusion bodies which activity was recovered after being renatured. Immunohistochemical results showed that ScFv retained almost the same antigen affinity and specificity as its parent monoclonal antibody.


CONCLUSIONS
Recombinant antibody ScFv is potential in both diagnostic and therapeutic application of human malignant tumors.
Wang
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Department of Oral and Maxillofacial Surgery, College of Stomatology, West China University of Medical Sciences, Chengdu, 610041, China.
Yang
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X
Yuan
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Q
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D000915:Antibody Affinity; D003001:Cloning, Molecular; D016228:Endothelial Growth Factors / Q000276:immunology*; D015870:Gene Expression; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D008222:Lymphokines / Q000276:immunology*; D051379:Mice; D009369:Neoplasms / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D000911:Antibodies, Monoclonal; D016228:Endothelial Growth Factors; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D008222:Lymphokines; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; C055966:immunoglobulin Fv