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

Objective To construct the eukaryotic expression plasrnid pcDNA4-rhHIF-1α of recombinant human hypoxia-inducible factor-1α.Methods Total RNA was isolated from blood 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-la,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.A1l sequences amplified by PCR were confirmed by complete sequencing.Correct sequences were cloned into the pcDNA4 vector.Results Recombinant eukaryotic expression plasmid pcDNA-rhHIF-la was gained.The target gene obtained by PCR amplification had the same molecular size as the estimate.It was indicated that the recombined pcDNA4 plasmid conrained 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.