Construction and identification of the eukaryotic expression plasmid for cyclooxygenase-2-specific siRNA

Objective To clone the recombinant eukaryotic expression plasmid of specific small interfering RNA(siRNA)against cyclooxygenase-2(COX-2)gene and to evaluate its activity of inhibiting the COX-2 expression in human colon cancer HT-29 cells.Methods The COX-2 siRNA template DNA sequence for short hairpin RNA(shRNA)was designed and synthetized.The annealed siRNA template was inserted into pGPH1-GFP-Neo plasmid.The recombinant plasmid(pGPH1-GFP-Neo-COX-2)was transformed into DH5α strain and identified by restrictive enzyme digestion and sequence analysis.The effect of the recombinant plasmid on COX-2 expression of human colon cancer HT-29 cells was detected by RT-PCR and Western blot.Results It was confirmed by restrictive enzyme digestion and sequence analysis that the recombinant plasmid was cloned and the aim sequence was obtained.COX-2 expression of HT-29 cells was inhibited at mRNA and protein levels 72 hours after transfected with the recombinant plasmid.Conclusion COX-2 siRNA expression plasmid pGPH1-GFP-Neo-COX-2 successfully constructed can inhibit the expression of COX-2 gene in human colon cancer HT-29 cells.