Construction and verification of eukaryotic expression plasmid carring human HO-1 gene

Objective:To construct and certificate eukaryotic expression plasmid carrying human HO-1 gene. Methods:By molecular biological technique,total RNA was extracted from human liver. After RT-PCR and TA cloning,the plasmid of pMD/19-HO-1 was successfully constructed. Then the plasmid was digested with restriction enzymes BamHⅠand EcoRⅠ. Then the human HO-1 gene fragment was cloned into plasmid pcDNA3.1 to construct the recombinant pcDNA3.1-HO-1 plasmid. pcDNA3.1-HO-1 was verified by restriction digestion and gene sequencing,The constructed plasmid were transfected into ECV304 cell line and certificated by Western blot analysis. Results:Identification of pcDNA3.1-HO-1 by restriction digestion and sequencing showed that the target gene was inserted into pcDNA3.1 correctly; the sequence of the target gene by DNA sequencing was identical with that of NCBI gene bank; the plasmid could increase the expression of human HO-1 obviously. Conclusion:pcDNA3.1-HO-1 was constructed successfully,which will help to study the effect of human HO-1 on vasculopathy in diabetes mellitus.