Research Of The Screen And Activity Of Relative Peptide Of Hypoxia-Inducible Factor-1

The translation factor hypoxia-inducible factor-1 (HIF-1) is a master regulator of the hypoxia response, and it occupies a central position in oxygen homeostasis in a wide range of organism. HIF-1 actives the expression of genes involved in angiogensis, energy metabolism, erythropoiesis, vasomotor control, apoptosis, and proliferation. It is crucial for drug development and biological research that HIF-1 activity was precisely modulated in cells, in tissues or in vivo. Because it is difficult for the protein passing through the cell membrane, we expect to find a oligopeptide for which can regulate HIF-1 activity, and provide a novel therapeutic strategies to ischemic disorders.1. The screen of relative peptide of HIF-1 a from a phage displayed library.The HIF-1 a mAb was used to react on a random sequenced peptide library of 12 amino acides. The dilution of HIF-1 a mAb and the strength of elution were increased from the first to the third round of selection. After the third round of biopanning, 10 clones of phages were randomly picked and their DNA inserts were sequenced. We obtained 7 sequences. Then 45 clones of phage were picked, and 22 positive clones of them were sequenced after Dot-ELISA test. The most repeated sequence isGPHHYWYHLRLPby adding up to two results. 2.Analysis and design of HIF-1 relative peptide.The sequences selected from 12-mer phage library were compared with HIF-1 a sequence in GenBank. The central sequenceof HIF-1 a relative peptide was determined according to the references. The selected sequences were used to design the analog peptides basing on the central sequence. The physical and chemical characterization of peptides were tested by Antheprot software, such as isoelectric point, molecular weight, half life, polarity. Finally, GLAPYIGPHH, one of eight candidate sequences was to be synthesized. 3. Research of the activity and mechanism of synthesized peptide.PC 12 cells were treated with different concentration of peptide to observe the effects on LDH, MDA and SOD after chemical hypoxia mimicked by CoCl2 8h. SOD activity was increased in a concentration dependent manner, but MDA level was declined. The peptide had no effects on LDH levels.In view of the results above, we studied the effects of peptide on the levels of HIF-1 a and VEGF protein by using of immunofluorescence technic. Laser Confocal Microscopy was used for observation of Fluorescence intensity, and the levels of HIF-1 a and VEGF protein were semi-quantitated. However, the peptide was found having no effects on the levels of HIF-1 a and VEGF protein no matter in normoxia or in hypoxia.