针对人SND1基因两个AUG的细胞应激分析

Analysis of Cellular Stress Response in Two AUG of Human SND1 Gene

目的针对人SND1基因2个蛋白翻译起始密码子AUG构建真核表达质粒pCMV-N-Flag-SND1-No1/2,并分析2个AUG在SND1应激颗粒形成中的作用。方法以SND1全长转录本为模板,PCR法扩增含BamHⅠ和EcoRⅠ酶切位点的目的基因SND1-No1/2,双酶切法分别酶切目的基因片段和线性pCMV-N-Flag,以T4-DNA连接酶将两者连接成pCMV-N-Flag-SND1-No1/2重组质粒,然后将构建的重组质粒转染入HeLa细胞内,以Western印迹法检测Flag标签(DYKDDDDK)与SND1-No1/2的融合表达,最后以细胞免疫荧光实验检测在氧化应激状态下Flag-SND1-No1/2融合蛋白与内源性SND1应激颗粒的胞内共定位情况。结果以单/双酶切及基因测序法鉴定构建的重组质粒无误,Western印迹结果检测到融合蛋白Flag-SND1-No1/2的表达;细胞免疫荧光结果显示Flag-SND1-No1/2均可与内源性SND1应激颗粒共定位。结论重组pCMV-N-Flag-SND1-No1/2质粒构建成功,SND1基因第1个AUG的缺失并不影响SND1应激颗粒的形成。

Objective To construct eukaryotic Flag （DYKDDDDK） expressing recombinant plasmids, pCMV-N-Flag-SND1-No1/2, which contain the coding sequence of human SND1-No1（from 1st AUG）or SND1-No2 （from 2nd AUG）, and perform the cellular localization analysis of Flag-tagged SND1-No1/2 under stress condition to study the function of the two AUG in the SND1 containing stress granules formation. Methods The gene fragments of SND1-No1/2 were amplified by PCR from the whole SND1 transcript and inserted into pCMV-N-Flag expressing vector through BamHI/EcoRI double en-zyme digestion and T4 DNA Ligase connection. The recombinant pCMV-N-Flag-SND1-No1/2 plasmids were transfected in-to HeLa cells and the expression of Flag-SND1-No1/2 fusion proteins was examined by Western blotting assay. Immunofluo-rescence assay was performed to detect the co-localization of Flag-SND1-No1/2 with endogenous SND1 granule. Results The pCMV-N-Flag-SND1-No1/2 were sequenced and digested correctly by restriction single/double enzyme. The Flag-tagged SND1-No1/2 fusion proteins were also detected in transfected HeLa cell by Western blotting assay. Both of them showed the co-localization with endogenous SND1 granule. Conclusion The recombinant eukaryotic plasmids of pCMV-N-Flag-SND1-No1/2 were constructed successfully and expressed effectively. The depletion of 1st AUG failed to af-fect the formation of SND1 containing stress granules.