干扰素调节因子3引导RNA真核表达载体的构建  

Construction of interferon regulatory factor 3 guided RNA eukaryotic expression vector

作　　者：鞠思雨 侯卓岩 王晰 姜生远 李佳起 吕海菲 刘文 张之勇 Ju Siyu;Hou Zhuoyan;Wang Xi;Jiang Shengyuan;Li Jiaqi;Lyu Haifei;Liu Wen;Zhang Zhiyong(Shandong First Medical University(Shandong Academy Of Medical Sciences),Jinan Shandong 250117,China)

机构地区：[1]山东第一医科大学临床与基础医学院,山东济南250117

出　　处：《中华临床实验室管理电子杂志》2022年第4期215-221,共7页Chinese Journal of Clinical Laboratory Management(Electronic Edition）

基　　金：山东省医药卫生科技发展计划项目(202002050626);国家自然科学基金青年科学基金(82100142);2021年度国家级大学生创新创业训练计划项目(202110439059)。

摘　　要：目的通过构建干扰素调节因子3(IRF3)gRNA真核表达载体,研究IRF3介导的转录非依赖性细胞凋亡的分子机制。方法通过分析IRF3的分子结构,在CHOPCHOP网站设计IRF3分子gRNA的序列,并在pLenti-U6-gRNA-PGK-Neo表达载体上ClaI和NheI限制性内切酶的位点处设计两对引物,通过重叠延伸PCR法扩增生成IRF3 gRNA的表达模块,通过TA克隆的方法构建到pGM-simple T克隆载体中,然后用双酶切的方法将IRF3 gRNA的表达模块亚克隆构建到表达载体中,通过PCR和Sanger测序的方法筛选并鉴定pLenti-U6-IRF3-gRNA表达载体。结果在IRF3的BH3样结构域和C端磷酸化结构域之间获得IRF3的gRNA序列。通过重叠延伸PCR扩增得到IRF3 gRNA的表达模块,通过TA克隆得到克隆载体pGM-simple T-IRF3-gRNA。通过双酶切的方法,将IRF3 gRNA表达模块亚克隆至pLenti-U6-gRNA-PGK-Neo真核表达载体,通过Sanger测序鉴定最终得到pLenti-U6-IRF3-gRNA表达载体。结论通过重叠延伸法成功构建pLenti-U6-IRF3-gRNA表达载体,为研究IRF3诱导细胞凋亡的转录非依赖性分子机制奠定了基础。Objective To investigate the molecular mechanism of interferon regulatory factor 3(IRF3)mediated transcription-independent apoptotic by constructing IRF3 gRNA eukaryotic expression vector.Methods IRF3's structure was analyzed and IRF3 gRNA was designed at CHOPCHOP,two pairs of primers were designed at ClaI and NheI site of pLenti-U6-gRNA-PGK-Neo expression vector.IRF3 gRNA expression module was amplified by gene splicing by overlap extension PCR(SOE PCR),and constructed in pGM-simple T clonal vector,and was subcloned in pLenti-U6-gRNA-PGK-Neo expression vector by dual digestion and IRF3 gRNA expression module was verified by bacteria PCR and Sanger sequencing.Results IRF3 gRNA was acquired between BH3 like domain and C terminal phosphorylation sites.IRF3 gRNA expression module was generated by SOE PCR.pGM-simple T-IRF3-gRNA clonal vector was constructed with IRF3 gRNA expression module by TA clone.pLenti-U6-IRF3-gRNA expression vector was constructed by dual digestion,and verified by Sanger sequencing.Conclusions The pLenti-U6-IRF3-gRNA expression vector was successfully constructed by SOE PCR,which laid a solid foundation on study of IRF3 mediated transcription-independent apoptotic mechanism.

关 键 词：干扰素调节因子3 重叠延伸PCR CRISPR-Cas9 引导RNA 

分 类 号：Q78[生物学—分子生物学]