Construction and function identification of rat carabin adenovirus interference vector

ObjectiveTo construct rat carabin adenovirus interference vectors (Ad-carabin-shRNA) with high titers.

MethodsThree pairs of carabin-shRNA oligonucleotide sequences were synthesized to construct ADV4-U6-CMV-carabin-shRNA shuttle plasmid.The ADV4-U6-CMV-carabin-shRNA shuttle plasmid and pHBAd-BHG plasmid were co-transfected into 293A cells to pack the Ad-carabin-shRNA.Viral titer was detected by whole-cell microlesion assay.H9C2 myocardial cells were infected with the Ad-carabin-shRNA, and the expression of carabin mRNA and protein was detected by real-time fluorescence quantitative PCR and Western blotting, respectively.

ResultsThree ADV4-U6-CMV-carabin-shRNA interference plasmids were successfully constructed, and the package of ADV4-U6-CMV-carabin-shRNA was successful.The titer of Ad-carabin-shRNA was 9×10⁸ TU/mL.The expression of carabin mRNA and protein was significantly downregulated in H9C2 myocardial cells after infected by Ad-carabin-shRNA-1 and Ad-carabin-shRNA-2 (P < 0.01).

ConclusionsThe construction of Ad-carabin-shRNA is successful, which exhibits interfering effect on carabin in H9C2 myocardial cells.