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目的:构建小鼠PNPLA7基因的敲降重组腺病毒,检测PNPLA7敲降重组腺病毒对小鼠肝脏PNPLA7蛋白的敲降效率及对肝脏甘油三酯含量的影响.方法:将对照siNC以及敲降PNPLA7的siRNA设计为shRNA并插入到pshuttle-CMV-silence穿梭质粒中,挑选克隆并测序鉴定,测序成功后将质粒进行PmeⅠ线性化,转入含有腺病毒骨架pAdeasy的BJ5183感受态细胞中进行同源重组,重组成功后的质粒经Pac Ⅰ线性化转染293A细胞进行腺病毒包装,得到Ad-shNC对照腺病毒以及Ad-shPNPLA7敲降腺病毒.通过尾静脉注射腺病毒7d后根据免疫印迹实验检测小鼠肝脏中PNPLA7敲降效率,同时利用Folch提脂方法提取并检测小鼠肝脏中甘油三酯含量.结果:成功获得对照腺病毒Ad-shNC以及敲降腺病毒Ad-shPNPLA7.通过尾静脉注射进行小鼠肝脏PNPLA7敲降,结果显示Ad-shPNPLA7腺病毒可成功降低PNPLA7的蛋白表达,敲降PNPLA7后引起肝脏甘油三酯含量上调.结论:小鼠PNPLA7的敲降腺病毒构建成功,敲降PNPLA7引起肝脏中甘油三酯含量升高.“,”Objective:To construct recombinant PNPLA7 shRNA adenovirus and check its knockdown efficiency in liver.Provide a new approach for further investigating the role of PNPLA7 on hepatic triglyceride metabolism.Methods:siNC and siPNPLA7 were designed and cloned into the p-shuttle-CMV-silence vector.After Sequencing identification,the plasmid was linearized by Pme Ⅰ and recombined with backbone pAdeasy in BJ5183 competent cells.The recombinant plasmid was linearized by Pac Ⅰ and transfected into 293A cells for packaging Ad-shPNPLA7 and Ad-shNC adenovirus.After 7 days of tail vein injection,Western blot was applied to determine the knockdown efficiency of pAdeasy-shPNPLA7 adenovirus in liver,and hepatic triglycerides content were measured,Results:The hepatic PNPLA7 protein levels were significantly decreased while the hepatic TAG levels were significantly increased in mice with tail vein injection of pAdeasy-shPNPLA7 adenovirus compared with control mice.Conclusion:The recombinant adenovirus of pAdeasyshPNPLA7 and pAdeasy-shNC were successfully constructed.The PNPLA7 may play a role in hepatic triglycerides metabolism.