Screening of human vascular endothelial growth factor (VEGF) receptor Flt-1 domain and study on its biological activity |
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Authors: | Li Ma Zhiqing Zhang Xiaoning Wang Dajun Sun Xiaoming Zhou Aijun Chen and Lihong Yao |
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Institution: | 1. Institute of Molecular Immunology,The First Military Medical University,Guangzhou 510515,China 2. National Laboratory of Molecular Virology and Genetic Engineering,Beijing 100052,China 3. Department of Vascular Surgery,The Third Teaching Hospital of Norman Bethune University of Medical Science,Changchun 130031,China |
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Abstract: | Four human vascular endothelial growth factor receptor Flt-1 cDNA fragments containing extracellular domain loops 2, 1–2, 2–3 and 1–3 respectively were amplified from human placental cDNA library by PCR and used for screening ligand binding domains by yeast two-hybrid system. The result showed that, not only loop 1–3, but also the smaller fragment loop 2–3 could bind to hVEGF165. Recombinant expression plasmids pPIC9K/Flt-1(1–3) and pPIC9K/Flt-1(2–3) were constructed and transformed toPichia. pastoris host strain GS115, cultured in flasks, and expressed under the induction of 1% methanol. The expressed product existed in supernatant in the form of soluble molecules and contained more than 60% of total protein after being induced for 4d. After being purified by CM-Sepharose FF and Sephacryl S-100 chromatography, its purity reached above 90%. Biological assayin vitro showed that the binding capacity of expressed soluble Flt-1 (2–3) to hVEGF165 and its inhibiting effect on the proliferation of human umbilical veins endothelial cells (HUVEC) stimulated with hVEGF165 were close to those of sFlt-1(1–3). Animal test showed that sFlt-1(2–3) could inhibit the formation of regenerate blood vessels stimulated with hVEGF165 significantly. |
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Keywords: | vascular endothelial growth factor(VEGF) receptor yeast two-hybrid Pichia pastoris gene expression |
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