Identification of a Neutralizing scFv Binding to Human Vascular Endothelial Growth Factor 165 (VEGF165) Using a Phage Display Antibody Library |
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Authors: | Zhihua Lin Peng Cao Huanzong Lei |
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Institution: | (1) Department of Biology, School of Chemistry and Life Sciences, Lishui University, Lishui, 323000, Zhejiang, People’s Republic of China;(2) Laboratory of Molecular Medicine, Jiangsu Province Institute of Traditional Chinese Medicine, 210028 Nanjing, People’s Republic of China |
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Abstract: | Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that plays a major role in angiogenesis. Alternative
splicing causes the production of several different isoforms (VEGF121, 145, 165, 183, 189, 206). VEGF is essential for tumor
angiogenesis, and several studies have correlated elevated VEGF levels with tumor stage, metastases, and progression. We now
report the isolation by phage display of human single-chain antibody fragment (scFv) anti-VEGF165. After four rounds of panning
against VEGF165, 40 out of 90 phage clones displayed VEGF165-binding activity. One of the positive clones, designated B8,
bound to VEGF165 with relatively high affinity and neutralized VEGF165 bioactivity in vitro. The B8 clone was expressed in
the soluble form in Escherichia coli HB2151 and purified by immobilized metal affinity chromatography. The purified scFv recognized VEGF165 with the K
D of 1.80 × 10−8 M without cross-reaction to VEGF121. In addition to binding, the purified scFv could does-dependently inhibit VEGF165-induced
human umbilical vein-derived endothelial cells proliferation. Together with its fully human mature, B8 scFv may have therapeutic
implications in therapy of angiogenesis-dependent diseases. |
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Keywords: | Phage display VEGF165 Single-chain antibody fragment (scFv) Angiogenesis-dependent diseases |
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