Identification And Characterization Of HIF-1α Form Pseudorasbora Parva

Pseudorasbora parva belongs to the gene Pseudorasbora (Cypriniformes:Cyprinidae), it is a various species distributed in rivers, lakes and ponds. Hypoxia-inducible factor la (HIF-1α) is a key transcription factor that controls a variety of cellular and systemic homeostatic responses to hypoxic stress. Pseudorasbora parva can survive in hypoxic, to study its hypoxia-inducible factor plays an important role in the regulation of genes.This study with Pseudorasbora parva contains5parts:1. The cloning and expression analysis of HIF-1α from Pseudorasbora parva. The cDNA was cloned using RT-PCR and RACE methods, and its nucleotide and protein sequences were analyzed with bioinformatics methods;2. The Pseudorasbora parva-actin gene as a internal control, using real-time fluorescence quantitative PCR (qRT-PCR) to detect HIF-la genes’mRNA expression level in hypoxic at8℃and18℃;3. The HIF-1α prokaryotic expression vector was constructed, and then transformed the vector to Escherichia coli BL21(DE3) in which we induced it expression, and then a fusion protein PET-17b-HIF-1α was purified to making antibody in New Zealand rabbit.4. Western blot of HIF-1α in the gill of fish in hypoxic was investigated using polyclonal antibody and the internal control of GAPDH.The results are offered as follows:1. The full-length cDNA of HIF-la is3753bp, containing a3’-untraslate region of1224bp and5’-untraslate region of204bp, which encords a deduced774amino acid peptide with a predicted molecular mass of85.83kD, the result of sequence alignments analysis showed that this amino acids shares92%identity with Ctenopharyngodon idella and protein shares97.8%identity with Ctenopharyngodon idella. The full-length cDNA sequence of the HIF-1α was submitted to GenBank and assigned the accession number FJ794604.2. qRT-PCR analysis was performed to quantify the relative expression levels of HIF-1α transcripts in low oxygen at8℃and18℃under different time. The results showed that HIF-la mRNA was expressed in all samples tested at different levels, and increased after being moved to hypoxic, and reached the hightest after6h, and go back to the unstimulated control level after24h, and remained constant after48h at8℃, but there was no significant change at18℃. 3. The HIF-1α prokaryotic expression vector was constructed with pET-17b, and the recombined plasmid was transfected into DE3. The result of SDS-PAGE showed that the molecular weight of the recombined protein was approximately26kD, expressed in supernatant. Using His affinity chromatography purified fusion protein, which was preparation for HIF-1α polyclonal anti-serum by immuning New Zealand rabbit. Western Blot showed that the anti-serum had good specificity.4. Western blot analysis revealed that HIF-1α protein was expressed in all samples tested at different levels, and increased after being moved to hypoxic, and reached the hightest after6h, and decreased after24h, but it was higher than the contol significantly, and decreased futher after48h.