Construction Of PHsa-miR16-shRNA Expression Vector And Determination Of Its Expression Activity

Objective1. To construct the expression vector of the siRNA aimed to X gene of HBV;2. Connected the new expression vector with the eukaryotic expression vector pmiR-16, in order to construct a new expression vector with two promoters, named pHsa-miR16-shRNA;3. Transcript the constructs in the human liver cancer cell lines, and identify its bioactivity by molecular biological techniques;4. Searching the relationship between miRNA, siRNA and the replication of HBV by our new expression vector.Methods1. Design and synthesis a pair of oligo DNA, about 60nt, cloned it to pSuper between the designed restriction site, obtain the new construction named siR-1583. The constructed plasmid siR-1583 was identified by enzyme restriction and the sequencing result.2. Design one pair of primers: 5'-CGGGATCCATTCGAACGCTGACGT-3'(sence)and 5'-CGGAATTCAAAAAGCACTTCGCTTCACC-3'( antisence ) , then amplified the sequence containing H1 promoter and the sequence which could encoding the DNA of HBV X siRNA. Insert the PCR products into pmiR-16 by the enzyme restriction BamHâ… and EcoRâ… , the new clone named pHsa-miR16-shRNA which has two expression frames.3. Transfect pHsa-miR16-siRNA into HepG2.2.15 cells, detect the content of HBsAg and HBeAg of the cell supernatant by ELISA; detect mRNA and HBV DNA by fluorescence.Results1. The result of enzyme restriction digestion shows what we anticipated, and the sequencing result of pHsa-miR16-shRNA also supported that the siR-1583 encoding gene have been cloned into pmiR-16 successfully.2. The results of ELISA, RT-PCR, fluorescent quantitation also shows that: pHsa-miR16-shRNA which could express miR-16 and the shRNA aimed to HBV X genes can more effective to depress the content of HBsAg and HBeAg.Conclusions1. The sequence of siRNA aimed to HBV X gene have been cloned into the eukaryotic expression vector pSuper successfully;2. Construct pHsa-miR16-shRNA which could both express human miR-16 and the siRNA which aimed to HBV X gene successfully;3. From the searching for the relationship between pHsa-miR16-shRNA and replication of HBV, we found that some siRNA and miRNA both have the effection on the replication of HBV. The construction of pHsa-miR16-shRNA set the research basic on the future research of miRNA,siRNA and HBV replication, even in HCC.