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    程阔菊, 吴庆, 罗健华, 魏谭军, 周殿儒, 肖成, 黄河, 罗云, 王毅. 大鼠carabin腺病毒干扰载体构建及功能鉴定[J]. 蚌埠医科大学学报, 2021, 46(3): 291-294. DOI: 10.13898/j.cnki.issn.1000-2200.2021.03.003
    引用本文: 程阔菊, 吴庆, 罗健华, 魏谭军, 周殿儒, 肖成, 黄河, 罗云, 王毅. 大鼠carabin腺病毒干扰载体构建及功能鉴定[J]. 蚌埠医科大学学报, 2021, 46(3): 291-294. DOI: 10.13898/j.cnki.issn.1000-2200.2021.03.003
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    CHENG Kuo-ju, WU Qing, LUO Jian-hua, WEI Tan-jun, ZHOU Dian-ru, XIAO Cheng, HUANG He, LUO Yun, WANG Yi. Construction and function identification of rat carabin adenovirus interference vector[J]. Journal of Bengbu Medical University, 2021, 46(3): 291-294. DOI: 10.13898/j.cnki.issn.1000-2200.2021.03.003
    Citation: CHENG Kuo-ju, WU Qing, LUO Jian-hua, WEI Tan-jun, ZHOU Dian-ru, XIAO Cheng, HUANG He, LUO Yun, WANG Yi. Construction and function identification of rat carabin adenovirus interference vector[J]. Journal of Bengbu Medical University, 2021, 46(3): 291-294. DOI: 10.13898/j.cnki.issn.1000-2200.2021.03.003
    shu

    大鼠carabin腺病毒干扰载体构建及功能鉴定

    Construction and function identification of rat carabin adenovirus interference vector

      摘要
    • 摘要:
      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。

       

      Abstract:
      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.

       

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