Construction and identification of CA916798 eukaryotic expression vector for RNA interference

Objective To construct an eukaryotic expression vector encoding an shRNA targeting CA916798. Methods According to the CA916798 cDNA sequence in GenBank, 2 pairs of oligo nucleotides were designed and synthesized. After primer annealing, they were inserted into plasmid pGenSil-l to construct the shRNA eukaryotic expression vector. The recombinant plasmid were transformed into DH5α, and the positive strain were identified by enzyme digestion and sequence analysis. The recombinant vector were transfected into A549/CDDP cells with Lipofectamine 2000. Drug sensitivity and proliferation of the transfected cells were measured by MTT test. Results The pRNAi-CA916798 shRNA of recombinant plasmid was constructed successfully. The growth of A549/CDDP cells transfected by pRNAi-CA916798 shRNA was slowly than that of those with blank vector transfection after 1.0 μg/ml CDDP treatment (P0.01). Conclusion The recombinant vector is established and transfected into A549/CDDP cells successfully,and A549/CDDP cell with CA916798 shRNA transfection are obtained.