Construction And Evaluation Of The Eukaryotic Expression Vector Of Human MiR-122

Objective1. To construct the eukaryotic expression vector of miR-122 by molecular biological techniques;2. Express this new construct in HepG2 cells, identify its biological activity;3. Research the relationship between miR-122 and HBV using this new construction.Methods1. The sequence encoding the mature miR-122 was amplified by PCR from the human genome DNA extracted from hepG2 cells. With the designed restriction site, digest the eukaryotic express vector pSuper and the amplified sequence, then link them, obtain the new construction. The constructed plasmid pHas-m122 was identified by enzyme restriction and the sequencing result.2. GFP-122si and GFP-124si were given as a present by Prof. Peter Sarnow from the Department of Microbiology and Immunology of Stanford University. The mature miR-122 could bind the 3'ORF of GFP-122si, so it could inhibit the expression of GFP, so is the relationship between miR-124 and GFP-124si, so we could co-transfect GFP-122si and pHsa-m122 into HepG2 cells, with GFP-124 as a control. Detect the expression of GFP by fluorescence, flow cytometry and western blot, the decline of the expression of GFP can reflect the effect of pHsa-m122.3. After that, we transfect pHsa-m122 into HepG2.2.15 cells, detect HBsAg and HBeAg of the cell supernatant by ELISA over 48h.Results1. The result of enzyme restriction digestion shows what we anticipated, and the sequencing result also supported that the pro-sequence of miR-122 have been cloned into the eukaryotic expression vector pSuper successfully.2. The results of fluorescence, flow cytometry and western blot also shows the differences of the content of GFP between the samples transfected with or no pHsa-m122, suggests that pHsa-m122 can express the mature miR-122 with biologic activity.3. The content of HBsAg, HBeAg in the supernatant of HepG2.2.15 cells transfected with pHsa-m122 increased.Conclusion1. The pro-sequence of miR-122 have been cloned into the eukaryotic expression vector pSuper successfully;2. pHsa-m122 could express the mature miR-122 with biological activity. pHsa-m122, as the eukaryotic expression vector of miR-122, can set up the base of doing future research on miR-122 and its function on the replication of HBV and the progress of HCC.