DAX-1真核表达载体的构建及其活性的检测

The construction of DAX-1 eukaryotic expression vector and detection of its activity

目的克隆剂量敏感的性别反转-先天性肾上腺发育不良基因1(DAX-1)的cDNA,构建带HA多肽标签的DAX-1真核表达载体,并检测其在小鼠肾上腺皮质瘤细胞(Y-1)中的转录活性.方法应用RT-PCR法从人睾丸中扩增DAX-1,将测序正确的PCR产物克隆至3'端带HA多肽标签pcDNA3.1(+)真核表达载体中,以脂质体方式将该质粒或pcDNA3.1(+)-3'HA空载与类固醇激素灵敏调节蛋白(StAR)启动子荧光素酶报告基因载体共转染至Y-1细胞中,24 h后利用双荧光素酶报告系统检测DAX-1对StAR表达的影响.结果RT-PCR获取编码DAX-1基因的全序列cDNA,大小为1413bp;DNA测序和PCR方法证实DAX-1基因成功克隆到pcDNA3.1(+)-3'HA真核表达载体中;瞬时转染Y-1细胞,经报告基因检测结果显示DAX-1与StAR共转染组(实验组)的荧光素酶活性是空载组(对照组)的1/3.结论成功构建人DAX-1真核表达载体,并在Y-1细胞中初步活性分析得知所克隆的DAX-1存在转录活性,为进一步研究DAX-1功能及调控机制奠定实验基础.

Objective To clone the cDNA and construct the eukaryotic expression vector of DAX-1,which contained HA peptide tag,and the transcriptional activity were detected in Y-1 cell.Methods DAX-1 was amplfed from human testis by RT-PCR.The sequenced PCR products were cloned into pcDNA3.1(+)vector,which contained HA peptide tag in its 3'domain.The recombined plasmid was sequenced and co-transfected into Y-1 cells by lipofectamine TM2000 with the luciferase reporter gene vector containing StAR promoter regions.After 24 hours,cells were collected to detect the activity of luciferase.Results The 1413bp cDNA of DAX-1 was obtained by RT-PCR.The DAX-1 gene was successful cloned into the pcDNA3.1(+)-3'HA vector which was confirmed by DNA sequencing and PCR.The experiment of transient transfection showed that the the luciferase activity of the experimental group was 66.7%lower than that of the control group.Conclusion The eukaryotic expression vector of human DAX1 has been constructed sucssully.The analysis of activity in Y-1 cells indicates that the recombined vector of DAX-1 does have transcriptional activity.This result may lay the foundation for further study the regulation of DAX-1 expression.