ABSTRACT
On the basis of the origin comparison of known endothelial genesis inhibitors, a 417-bp cDNA fragment was amplified from umbilical cord by RT-PCR and cloned into the expression vector pPIC9, followed by transformation into Pichia pastoris GS115. The resulted yeast was induced with methanol to express recombinant protein. The resulted protein was purified from culture broth and designated as EDI-8t. The in vitro study showed that EDI-8t, originated from collagen VIII, could specifically inhibit the growth and migration of bovine aortic endothelial cells (BAEC) stimulated by basic fibroblast growth factor (bFGF). The protein also exhibited the activity to cause cell apoptosis. In vivo EDI-8t showed the identical activity comparing with endostatin to inhibit the growth of liver tumor transplanted into nude mice. Interestingly, EDI-8t showed higher activity than endostatin to inhibit tumor growth in metastatic model of melanoma mice.
Subject(s)
Animals , Cattle , Humans , Mice , Amino Acid Sequence , Angiogenesis Inhibitors , Genetics , Antineoplastic Agents , Pharmacology , Base Sequence , Cells, Cultured , Collagen Type VIII , Chemistry , Genetics , Endothelium, Vascular , Metabolism , Genetic Vectors , Genetics , Human Umbilical Vein Endothelial Cells , Chemistry , Mice, Nude , Molecular Sequence Data , Pichia , Genetics , Metabolism , Recombinant Proteins , Genetics , PharmacologyABSTRACT
Angiogenesis-related diseases involving EGF include acherosclerotic plaques, haemangioma, angiofibroma, tumor growth and arthritis. EGF may serve as a drug target and its antagonists may have important clinical applications.Peptide phage display libraries have been successfully applied in areas of finding ligands for enzymes, receptors, and many other molecules. A pⅧ-based peptide phage display library was panned with the cytokine EGF and several EGF-binding clones were selected based on ELISA and micropanning assays. The selected EGF-binders from peptide phage display library may be utilized in affinity chromatography in EGF downstream processing and even act as potential antagonists of EGF if their affinity is further improved through secondary library strategy.