内蒙古绒山羊TNFRSF1A基因shRNA表达载体的构建及鉴定

Construction and Identification of shRNA Expression Vector of TNFRSF1A Gene in Inner Mongolia Cashmere Goat

通过设计和合成TNFRSF1A基因的3条靶向shRNA,并分别插入到pSGU6/GFP/Neo载体中,成功构建出了3个干扰载体,通过脂质体介导法将干扰载体成功转入到内蒙古绒山羊胎儿成纤维细胞中,并利用RealTimePCR检测TNFRSF1A基因的表达量。由结果可知,TNFRSF1A基因的干扰载体经测序鉴定证明构建成功,干扰载体转染内蒙古绒山羊胎儿成纤维细胞24h后可见绿色荧光表达,当到达48h时,转染效率达到最大,shRNA-1、shRNA-2、shRNA-3三组中TNFRSF1A的mRNA表达水平均低于转染空载体的NC组,且shRNA2对TNFRSF1A的mRNA表达具有最佳的干扰效果。综上所述,该研究成功构建了TNFRSF1A干扰载体,为进一步研究绒山羊TNFRSF1A基因在毛囊生长与周期调控的作用奠定基础。

In this study,3 target shRNA of TNFRSF1A gene were designed and synthesized and inserted into the pSGU6/GFP/Neo vector respectively,and 3 recombinant plasmid interference vectors were constructed.The recombinant plasmid was transferred into fibroblasts from Inner Mongolia cashmere goat by liposome mediated method,and detection of TNFRSF1A gene expression by Real time PCR.The results showed that the shRNA recombinant plasmid of TNFRSF1A gene was successfully constructed by sequencing.The recombinant plasmid was transfected into Inner Mongolia cashmere goat fibroblasts for 24 hours,and the green fluorescent expression was observed.The transfection efficiency reached the maximum after 48 h,the mRNA expression level of TNFRSF1A in shRNA-1,shRNA-2 and shRNA-3 was lower than that in NC group transfected with empty vector,and shRNA2 had the best interference effect on mRNA expression of TNFRSF1A.In summary,this study successfully constructed the TNFRSF1A interference vector,which layed a foundation for further study on the role of TNFRSF1A gene in the growth and regulation of hair follicles.