大鼠STAT3特异性shRNA慢病毒载体的构建与鉴定

目的在已经成功构建4个针对STAT3的shRNA质粒表达载体的基础上,筛选出干扰效果最佳者进行慢病毒包装并鉴定。方法将STAT3的干扰质粒shRNA1、shRNA2、shRNA3、shRNA4及Control1(空载体对照),Control2(无效shRNA对照)分别转染目的细胞(大鼠肾小球系膜细胞),48h后收集细胞提取mRNA,以RT-PCR检测各组分STAT3表达量。对于筛选所得干扰效果最佳的质粒,采用pPACKH1TM慢病毒载体系统进行病毒包装。利用绿色荧光蛋白作为报告基因,对感染效率及病毒滴度进行检测。结果 RT-PCR检测结果显示,shRNA3样品受干扰的效果最佳。成功构建了慢病毒载体Lenti-shRNA3,共转染293T细胞24h、48h,荧光显微镜下可见强绿色荧光,细胞生长状态良好,表明病毒包装成功。转染HT1080细胞,收集慢病毒液,测定病毒滴度为3.34×108ifu/ml,适合感染目的细胞。结论成功构建了STAT3基因的shRNA慢病毒表达载体,为进一步应用奠定了基础。

Construction and identification of a recombinant lentivirus harboring shrna targeting rat STAT3 gene

Objective Based on the 4 plasmids expressing shRNA targeting STAT3 successfully constructed earlier,to screen the one with best interference effect,and conduct lentivirus packing and identification.Methods The target cells(rat mesangial cells) were transfected with the 4 STAT3 shRNA plasmids: shRNA1,shRNA2,shRNA3,shRNA4 and the empty vector control(Control1),invalid shRNA control(Control2).mRNA was extracted and expression of STAT3 was measured by RT-PCR after 24 hours of incubation.Lentivirus packing was done on the one with best interference effect using pPACKH1TM packaging plasmids.The titer and infection efficiency of the recombinant lentivirus were determined according to the expression of the reporter gene enhanced green fluorescent protein(EGFP) uner fluorescent microscope.Results RT-PCR results showed that shRNA3 was the most effective in STAT3 interference.A recombinant lentivirus Lenti-shRNA3 was successfully constructed and co-transfected into 293T cells.24h and 48h later,green fluorescence were detected under inverted fluorescence microscope.Then Lenti-shRNA3 was tranfected into HT1080 cells,the supernatant was collected and the titer of the concerned virus was 3.34×108ifu/ml.Conclusion Recombinant lentivirus plasmid of RNA interference targeting rat STAT3 gene was successfully constructed,which could suppress STAT3 expression with high efficiency,and can be used in further study.