Construction of eukaryotic expression vectors of siRNA against TNF-α gene and their silencing effects on TNF-α and TGF-β1 in A549 cells

Aim: To clone the eukaryotic expression vectors of small interfering RNA (siRNA) against TNF-α gene and to evaluate their silencing effects on TNF-α and TGF-β1 in A549 cells.Methods:According to TNF-α gene (NM_000594) sequence of GenBank, using RNAi Designer software to design the siRNA specific target sequence (siTNF-α) and the unrelated control sequence (siCon) for TNF-α. The annealing products were recombined into pRNAT-U6.1, PCR and DNA sequencing confirmed the insertion sequence. The recombinant plasmids (pRNAT-U6.1-siTNF-α,pRNAT-U6.1-siCon) were transfected to the A549 cells with lipofectamine,and the expressions of TNF-α, TGF-β1 mRNA and protein in cells detected by Real time PCR and Western blot methods.Results:PCR and DNA sequencing confirmed the vectors were consistent;the expressions of TNF-α, TGF-β1 mRNA and protein in the cells transfected pRNAT-U6.1-siTNF-α were markedly lower than the other three controls(FmRNA=478.663,4.081;Fprotein=123.420,6.312,all P0.05).Conclusion:The TNF-α siRNA eukaryotic expression vector is constructed successfully,which could silence the expression of TNF-α in A549,as well as the expression of TGF-β1.