Construction and identification of eukaryotic expression plasmid pcDNA4/rhHIF-1α

[Objective] To construct the eukaryotic expression plasrnid pcDNA4-rhHIF-1α of recombinant human hypoxia-inducible factor-1α(HIF-1α).[Methods] Total RNA was isolated from blood cells and cDNA was constructed by reverse transcriptional PCR method.The two oligomers of primers were synthesized based on the reported sequences of HIF-lα,the cDNA prepared as above was used as the template,and the HIF-1α fragment A and fragment B 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 pcDNA4/rhHIF-lα was gained.The target gene obtained by 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 of HIF-1α gene clone,expression and antibody preparation.