水牛SIRT6基因shRNA干扰慢病毒载体构建与验证

Construction and Verification of Buffalo SIRT6 Gene shRNA Interference Lentiviral Vector

本研究旨在构建SIRT6基因的RNA干扰慢病毒载体,并在水牛成纤维细胞中验证载体的有效性。设计合成2条特异性水牛SIRT6基因的shRNA序列,分别将其克隆至Psi-LVRU6GP慢病毒干扰载体上,重组质粒通过琼脂糖凝胶电泳及测序鉴定。将重组质粒与慢病毒包装载体NRF、VSVG共转染293T细胞,荧光显微镜下检查转染效果。转染60 h后收集病毒悬液,并感染水牛胎儿成纤维细胞(BFF),qRT-PCR检测干扰效果。结果显示:成功构建了2个RNA干扰SIRT6基因表达的慢病毒重组质粒;经慢病毒包装感染水牛成纤维细胞后检测SIRT6 mRNA表达水平,干扰载体Psi-sh1-LVRU6GP、Psi-sh2-LVRU6GP均能有效下调SIRT6的表达,与质粒对照组相比差异显著,Psi-sh2-LVRU6GP的干扰效果最好。SIRT6基因shRNA干扰慢病毒载体的成功构建为研究SIRT6基因对水牛细胞衰老的影响及其机制奠定基础。

This study aimed to construct a RNA interference lentiviral vector of the SIRT6 gene and validate the vector in buffalo fibroblasts.Design and synthesize two specific shRNA sequences of buffalo SIRT6 gene.They were cloned into Psi-LVRU6GP lentivirus interfering vector,respectively.The recombinant plasmid was identified by agarose gel electrophoresis and sequencing.The recombinant plasmid was co-transfected into 293T cells with the lentiviral packaging vector NRF and VSVG,and the transfection effect was examined under a fluorescence microscope.After 60 hours of transfection,the virus suspension was collected and infected with buffalo fetal fibroblasts(BFF),and the interference effect was detected by qRT-PCR.The results show that two recombinant lentivirus plasmids with RNA interfering SIRT6 gene expression were successfully constructed.SIRT6 mRNA expression levels were detected after infection of buffalo fibroblasts by lentiviral packaging.The interference vectors Psi-sh1-LVRU6GP and Psi-sh2-LVRU6GP were able to effectively down-regulate the expression of SIRT6,which was significantly different from the plasmid control group(P<0.05).Psi-sh2-LVRU6GP has the best interference effect.The successful construction of SIRT6 gene shRNA interference lentiviral vector lays a foundation for studying the effect of SIRT6 gene on buffalo cell senescence and its mechanism.