Construction and Identification of Eukaryotic Expression Vector of miR-16

Objective To construct eukaryotic expression vector of miR-16.Methods miR-16 precursor sequence was amplified from the human genomic DNA,and was introduced into the restriction sites and polyT transcription terminate sequence.The target gene was inserted to the digested pSuper siRNA expression vector.Restriction digestion,ligation and sequencing were performed to evaluate the recombinant.Then pXF3H-HBs expression vector containing miR-16 complementary sequence was constructed the report plasmid-HBs were constructed.The pmiR-16 and report vector pXF3H-HBs were co-transfected into HepG2 cells to detect the biological activity of pmiR-16.Results The miR-16 expression vector and its report vector pXF3H-HBs were successfully constructed.pmiR-16 and pXF3H-HBs were co-transfected into HepG2 cell line,HBsAg gene expression,as compared with the control group,was decreased significantly.Co-transfection confirmed the biological activity of pmiR-16.The expression of HBsAg was obviously decreased.Conclusion pmiR-16 expression vector has been successfully constructed.