Construction,Expression and Biological Activity Studies of a Bivalent Single Chain Variable Fragment(BsFv)against Vascular Endothelial Growth Factor Receptor 2

2011, Vol. 31

Issue (8): 1-6 DOI:

Construction,Expression and Biological Activity Studies of a Bivalent Single Chain Variable Fragment(BsFv)against Vascular Endothelial Growth Factor Receptor 2

DING Li, WANG Xiu-yun, QI Hai-di, LI Hai-xin, ZHOU Ya-qiong, CHEN Yao-zu, ZHANG Juan, WANG Min

School of Life Science & Technology,China Pharmaceutical University,Nanjing 210009,China

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Abstract

It was focused on that,the binding efficiency of Vascular Endothelial Growth Factor(VEGF)and Vascular Endothelial Growth Factor Receptor-2(VEGFR-2)competitively inhibited by bivalent single-chain Fv(BsFv),and BsFv's construction,expression and bioactivity identification. PCR primers were designed to introduce interlinker(G₄S)₃ between two scFv AK404 fragments,which were ligated with vector pET22b and transformed into E.coli BL21(DE3).The expression product was purified by IMAC and renatured by dialysis. The KDR-binding activity was determined by Bio-Layer Interferometry(BLI)and its influence on the propagation of HUVEC cell induced by VEGF. Bsfv was successfully constructed,and expressed as inclusion body after having been induced by 1 mmol/L IPTG for 6h at 37 ℃. After purification by IMAC and renaturation of the denatured recombinant protein,the BsFv with bioactivity and 90% purity was obtained. It was characterized to be target protein by Western blotting. The result of binding experiment in vitro based on BLI had proved that BsFv has higher binding ability to KDR3 than AK404. Additionally,BsFv was shown to inhibit the HUVEC cell's proliferation in a concentration-dependent manner,and the inhibition rate was relatively higher than that of AK404. All the results indicated that BsFv have higher anti-KDR activity compared with scFv,which provided a potential candidate drug in further research on its application on antiangiogenic oncotherapy and tumor diagnostics.

Key words： Vascular endothelial growth factor receptor 2 Bivalent single chain Fv Bio-layer interferometry Antiangiogenic oncotherapy

Received: 13 April 2011 Published: 25 August 2011

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Cite this article:

DING Li, WANG Xiu-yun, QI Hai-di, LI Hai-xin, ZHOU Ya-qiong, CHEN Yao-zu, ZHANG Juan, WANG Min. Construction,Expression and Biological Activity Studies of a Bivalent Single Chain Variable Fragment(BsFv)against Vascular Endothelial Growth Factor Receptor 2. China Biotechnology, 2011, 31(8): 1-6.

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https://manu60.magtech.com.cn/biotech/ OR https://manu60.magtech.com.cn/biotech/Y2011/V31/I8/1

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