猪Sirt2慢病毒干扰载体的构建

Construction of a recombinant lentivirus harboring small interfering RNA targeting porcine Sirt2 gene

为了研究猪Sirt2基因在脂肪细胞增殖和分化中的作用,试验针对Sirt2基因设计并合成了3对siRNA序列,经退火、酶切后连接于慢病毒表达载体LentiH1上,然后与慢病毒包装质粒混合共转染293T细胞,48 h后收集细胞,浓缩病毒,并检测病毒效价和感染效率。结果表明:3个shRNA慢病毒干扰载体经包装浓缩后获得的病毒效价均为1×107tu/mL;感染猪脂肪细胞后,Western-blot检测表明Sirt2的蛋白表达受到显著抑制,其中1号病毒载体干扰效果最为明显。说明携带靶向Sirt2基因的RNAi慢病毒载体构建成功。

To study the role of sirt2 gene in the proliferation and differentiation of porcine adipocyte,three pairs of small interfering RNAs（siRNAs） targeting sirt2 were designed,synthesized and linked to lentivirus vector LentiH1 to construct the lentivirus shuttle plasmids.After sequencing,the three lentivirus shuttle plasmids were transfected into 293T cells in the presence of packaging plasmids.Forty-eight hours after transfection,the supernatant was collected and the titer and infection efficiency of the recombinant lentivirus were determined according to the expression of the reporter gene enhanced green fluorescent protein（EGFP） under fluorescent microscope.DNA sequencing demonstrated that the sirt2 shRNAs were successfully cloned into the lentivirus vector LentiH1,a high titer of the virus was obtained.After infection of adipocyte,Western-blot showed that the expression of sirt2 was suppressed,the interference effect of No.1 viral vector was most obvious among the vectors.It concludes that the recombinant lentivirus containing shRNAi target sirt2 gene has been successfully constructed,the differentiation of porcine adipocyte has been suppressed after infected by lentivirus.