Construction of recombinant eukaryotic expression vector pEGFPCI-HIF-1α and its expression in eukaryotic cells PC12 / 中华创伤杂志

ABSTRACT

Objective To construct the eukaryotic expression vector with the mutant of hypoxia inducible factor 1α (HIF-1α) that could over express nonhypoxic dependent HIF-1α and investigate its expression in eukaryotic cells PC12. Methods The coding sequence of HIF-1α was amplified from total RNA from the injured spinal cord of rats by means of RT-PCR. Then, overlapping extension PCR was employed to obtain one complete sequence encoding HIF-1α mutant without oxygen-sensitive degradation domain (ODD). By using gene recombination technique, the gene HIF-1α△ODD was inserted into eukaryotic expression vector _pEGFPC1 that contained a reporter gene of enhanced green fluorescent protein. The recombinant plasmid was successfully constructed and transfected into the neural cell line PC12. The expression of HIF-1α△ODD was analyzed by fluorescent observation and Western blot. Results A 2kb gene fragment encoding H1F-1α△ODD was successfully obtained by overlapping extension PCR. On this basis, the recombinant plasmid _pEGFPC1-HIF-1 α△ODD was constructed and testified with restriction enzyme digestion and DNA sequencing. After this recombinant plasmid was transfected into the neural cell line PCI2, both fluorescent observation and Western blot demonstrated that HIF-1α△ODD was over expressed under nonhypoxia conditions. Conclusion The eukaryotic expression vector _pEGFPC1-HIF-1α△ODD is successfully constructed and HIF-1α△ODD over expresses in transfected PCI2 cell lines.