Construction and identification of eukaryotic expression plasmid of recombinant human hypoxia-inducible factor-1? / 吉林大学学报(医学版)

ABSTRACT

Objective To construct the eukaryotic expression plasmid of recombinant human hypoxia-inducible factor-1?(HIF-1?) and get ready for the coloning and expressing of HIF-1? gene. Methods The total RNA was isolated from blood cells,and cDNA library was constructed by reverse transcription PCR method.PIREGFP containing EGFP fragment and cDNA were used as templet,the three designed primers (EGFP-linker,HIF-1? upstream and HIF-? downstream)were put into,after amplification the target gene fragment was inserted into the shuttle vector T,and transfected with E.coli DH5?,the bacterial colonies containing recombinant plasmids were identified by LB/KANA-agar plate,and the recombinant plasmids were extracted and purified.All sequences amplified by PCR were confirmed by complete sequencing.The correct sequences were cloned into the pVAX1 vector.Results The amplified fragments were about 870,1 199,672 bp by PCR,they were inserted into the shuttle vector T and sequenced. The result of sequencing was identical with that provided by GenBank.The final plasmid about 1 800 bp was obtained by the identification of enzyme digestion and it had same molecular size as expected.Conclusion The eukaryotic expression plasmid pVAX1-EGFP-linker-HIF-1? of recombinant human HIF-1? is successfully constructed.