IRE1结构域分析及截短型载体的构建和表达

目的 克隆IRE1基因,根据IRE1基因不同生物学功能的4个结构域构建截短型真核表达载体,并用生物信息学方法对其蛋白产物进行分析.方法 应用PCR重组技术,以pCMV-IRE1为模板扩增IRE1全长及4个截短型片段,利用DNA重组技术将片段定向插入到真核表达载体pcDNA3.1（-）中,经酶切及测序鉴定后,免疫印迹检测各重组载体在细胞中的表达,并利用SWISS-MODEL在线软件预测其蛋白结构.结果 酶切鉴定及Western 印迹结果表明成功构建了IRE1全长及每一种截短型片段的真核表达载体：pcDNA3.1（-）-IRE1（pIRE1）,pcDNA3.1（-）-IRE1-NLDP（pNLD）,pcDNA3.1（-）-IRE1-KinaseP（pKinase）,pcDNA3.1（-）-IRE1-R＋L（pR＋L）,pcDNA3.1（-）-IRE1-RNase（pRNase）.结论 IRE1及其截短型真核表达载体的成功构建和表达,为进一步研究IRE1各个结构域的生物学功能奠定了基础.

Analysis of IRE1 Domain and Construction of Truncated IRE1 Expression Vector

Objective To clone IRE1 gene and construct the eukaryotic expression vector containing four different structural fragments of IRE1 gene. Methods The four fragments were amplified from pCMV-IREI vector and inserted into the eukaryotic expression vector pcDNA3. 1 （ - ） respectively by PCR. After double-enzyme digestion check and sequencing, the recombinant plasmids were transfected into LO2 cells. Expression of each protein was detected by Western blot. Protein structures of the 4 proteins were predicted by the software SWISS-MODEL online. Results The recombinant plasmids of full-length and four fragments were constructed successfully, including pIRE1, pNLD, pKinase, pR ＋ L and pRNase. Conclusion The eukaryotic expression vectors that express full IRE1 and its fragments are applicable for further biological function research of IRE1.