Zyxin基因真核表达载体的构建及检测

Construction and detection of eukaryotic expression vector containing Zyxin gene

为了研究联蛋白基因(Zyxin)功能,对Zyxin基因进行了真核表达载体的构建,并对其进行鉴定。用全基因合成的方法得到Zyxin目的基因,将目的基因与pEX-6载体分别进行酶切,然后进行连接,连接产物转化到感受态大肠杆菌。挑取克隆,经质粒DNA抽提和酶切,琼脂糖凝胶电泳和测序,对克隆片段进行鉴定。将重组质粒pEX-6-Zyxin瞬时转染293T细胞。结果显示:电泳、双酶切和测序结果与预期结果相符。转染48 h后观察到红色荧光,用实时荧光定量PCR检测转染后48 h和72 h Zyxin基因mRNA水平有较高表达。表明成功构建了鸡Zyxin真核表达载体,为进一步研究该基因功能奠定了基础。

To study the function of Zyxin gene, the eukaryotic expression vector of Zyxin gene were established and identified. All gene synthesis methods were used to achieve the complete CDS sequence of Zyxin gene. The Zyxin and vector pEX-6 were separately excised. The excised products were connected and then transformed into competent Escherichia coli. The positive clones were selected, from which plasmid DNA was abstracted and identified by sequencing. The recombinant plasmid was instantaneously transfected into 293T cells. The results of electrophoresis, double digestion and sequencing were in accordance with expectations. The red fluorescence was observed 48 h posttransfection. High level expression of Zyxin was detected by real-time PCR 48 and 72 h post-transfection. These results indicated that pEX-6-Zyxin expression vector had been successfully constructed.