IRE1结构域分析及截短型载体的构建和表达  

Analysis of IRE1 Domain and Construction of Truncated IRE1 Expression Vector

作　　者：刘平[1,2] 李祥柱[2] 赵文君[2] 刘艳娜[1] 周菁华[1] 郭风劲[1,2] 

机构地区：[1]重庆医科大学细胞生物学及遗传学教研室,重庆市400016 [2]重庆医科大学分子医学与肿瘤研究中心,重庆市400016

出　　处：《医学分子生物学杂志》2011年第2期108-113,共6页Journal of Medical Molecular Biology

基　　金：国家自然科学基金（No.31040019）,教育部留学人员基金（No.2009-1590）

摘　　要：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.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.

关 键 词：内质网应激 需肌醇酶 结构域 载体构建 

分 类 号：Q78[生物学—分子生物学]