牛DKK2基因pcDNA3.1表达载体的构建

Construction of pcDNA3.1 Expression Vector of Cow DKK2 Gene

DKK2基因是Wnt信号通路的关键抑制因子,在胚胎发育、颗粒细胞生成等过程中发挥重要作用。扩增了牛DKK2基因CDS序列,构建了DKK2基因pc DNA3.1真核表达载体;为进一步验证载体在细胞内的表达效果,将表达质粒瞬时转染CHO细胞,利用q RT-PCR和Western blot检测DKK2的表达情况。结果表明,在CHO细胞中转染DKK2基因的真核表达载体质粒,其m RNA和蛋白质表达量均显著上升,表明已成功构建了DKK2基因表达载体,为下一步开展DKK2基因功能研究奠定了基础。

DKK2 gene is the key inhibitory factor of the Wnt signaling pathways, and plays an important role in the process of embryonic development, granular cell generation and so on. This study amplified the CDS sequence of cow DKK2 gene and built its pcDNA3.＇I eukaryotic,then verified the correctness by enzyme digestion and sequencing. In order to further verified the expressing effect of this expression vector in cells, the vector was transfected into CHO cells transiently, then DKK2 expression was detected by using qRT-PCR and Western blot. The results showed that the mRNA and protein expressions of DKK2 gene were significantly increased in CHO cells, and indicated the pcDNA3.1 of DKK2 gene had been successfully constructed. This laid the foundation of the next step of DKK2 gene function research.