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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。
AIM To construct eukaryotic expression vector pCMV-N-Flag-SND1-No1 / 2 targeting at two SNPs of human SND1 gene and analyze the role of two AUGs in SND1-induced stress granule formation. Methods SND1 full-length transcript was used as a template. The target gene SND1-No1 / 2 containing BamHⅠ and EcoRⅠ restriction sites was amplified by PCR. The target gene fragment and linear pCMV-N-Flag were digested by double enzyme digestion T4-DNA ligase to construct pCMV-N-Flag-SND1-No1 / 2 recombinant plasmids. The constructed recombinant plasmids were transfected into HeLa cells. Western blotting was used to detect the relationship between Flag tag (DYKDDDDK) and SND1- No1 / 2. Finally, the intracellular co-localization of Flag-SND1-No1 / 2 fusion protein and endogenous SND1 stress particles under oxidative stress was detected by immunofluorescence staining. Results The constructed recombinant plasmids were identified by single / double enzyme digestion and gene sequencing. Flag-SND1-No1 / 2 fusion protein was detected by Western blotting. The results of immunofluorescence showed that Flag-SND1-No1 / 2 Endogenous SND1 stress particles co-localization. Conclusion The recombinant plasmid pCMV-N-Flag-SND1-No1 / 2 was successfully constructed. The deletion of the first AUG of SND1 gene did not affect the formation of SND1-stimulated granules.