大鼠carabin腺病毒干扰载体构建及功能鉴定

目的构建高滴度大鼠carabin腺病毒干扰载体（Ad-carabin-shRNA）。方法设计3对carabin-shRNA寡核苷酸序列，分别构建3个ADV4-U6-CMV-carabin-shRNA腺病毒穿梭质粒，将构建好的ADV4-U6-CMV-carabin-shRNA和pHBAd-BHG质粒共转染293A细胞，以包装Ad-carabin-shRNA，采用微量全细胞病变法检测病毒滴度。Ad-carabin-shRNA感染H9C2心肌细胞，实时荧光定量PCR和Western blotting检测carabin mRNA及蛋白表达。结果3个ADV4-U6-CMV-carabin-shRNA干扰质粒构建成功，相对应的Ad-carabin-shRNA包装顺利，病毒滴度均为9×108 TU/mL。感染H9C2心肌细胞后，Ad-carabin-shRNA-1和Ad-carabin-shRNA-2均有显著干扰效果，carabin mRNA和蛋白表达明显下调（P < 0.01）。结论Ad-carabin-shRNA构建成功，在心肌细胞中具有干扰carabin的效应。

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.