Construction And Identification Of SiRNA Eukaryotic Expression Vectors Targeting On TGF-β1,TIMP-1,TIMP-2 Genes

Objective: To construct the siRNA eukaryotic expression vectors targeting on TGF-β1,TIMP-1 and TIMP-2 genes and observe their effects on liver fibrosis.Methods:Two pairs of 21bp reverse repeated sequences targeting on TGF-β1,TIMP-1 and TIMP-2 mRNA spaced by 9bp nucleotides were synthesized respectively. A generic unrelated control plasmid was synthesized at the same time. The complement double strands was formed by annealing and inserted into plasmid pGenesil-1 to generate eukaryotic expression plasmids. The recombinant plasmids were transformed into JM109 strain, and the recombinant plasmids extracted were identified by restriction enzyme and sequence analysis. In vitro transfer of the HSC-T6 cell culture,RT-PCR method to detect gene inhibition efficiency.Results:Recombinant plasmids were completely coincided with the designs by the restriction enzyme and the sequence analysis. Observed under fluorescence microscope, we can see a large number of cells with fluorescence. RT-PCR results showed that six of the siRNA expression vector-specific have inhibitory effects, in which psi-TF1-1, psi-TIMP1-1, psi-TIMP2-2 are more efficient.Conclusion: The recombinant plasmids targeting on TGF-β1,TIMP-1 and TIMP-2 genes were successfully constructed. The recombinant plasmids transfected into HSC-T6 cells in vitro,compared to untransfected group,recombinant expression plasmids resulted in reduction of TGF-β1,TIMP-1 and TIMP-2mRNA .