Construction and identification of eukaryotic expression plasmid pcDNA4-rhHIF-1α expressing recombinant human hypoxia-inducible factor-1α

Objective To construct the eukaryotic expression plasmid pcDNA4-rhHIF-1α of recombinant human hypoxia-inducible factor-1α.Methods Total RNA was isolated from Hela cells,and cDNA library was constructed by reverse transcriptional PCR method.The two oligomers of primers were synthesized based on the reported sequences of HIF-1α,the cDNA prepared as above was used as the template,and the HIF-1α fragment A(amino acids 1～390) and fragment B(amino acids 786～826) were constructed and inserted into the shuttle vector T.Transformation of E.Coli JM109 with recombinant plasmids and identification of bacterial colonies containing recombinant plasmids by LB-agar plate containing 100 μg/ml of amplicillin were conducted,and recombinant plasmids were extracted and purified.All sequences amplified by PCR were confirmed by complete sequencing.Correct sequences were cloned into the pcDNA4 vector.Results Recombinant eukaryotic expression plasmid pcDNA-rhHIF-1α was gained.The target gene obtained PCR amplification had the same molecular size as predicted.It was indicated that recombined pcDNA4 plasmid contained correct recombinant human HIF-1α sequences. Conclusion The pcDNA4 is high expressing eukaryotic vector,and can express the target gene in high level.The pcDNA4-rhHIF-1α plasmid is constructed successfully and will be useful for further research.