干扰素调节因子3引导RNA真核表达载体的构建

doi:10.3877/cma.j.issn.2095-5820.2022.04.005

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。

关键词: 干扰素调节因子3, 重叠延伸PCR, CRISPR-Cas9, 引导RNA

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.

Key words: Interferon regulatory factor 3, SOE PCR, CRISPR-Cas9, gRNA

图1 干扰素调节因子3的蛋白质结构与IRF3 gRNA的位置设计注：紫色碱基序列为IRF3 gRNA序列，红色碱基显示IRF3 gRNA的PAM序列，黑色对应IRF3 C端的碱基序列和氨基酸序列，含有丝氨酸，苏氨酸磷酸化受体群，蓝色氨基酸序列显示IRF3 IAD结构域中的SH3样结构域。IRF3 gRNA的序列在IRF3基因组中位于BH3样结构域与C端磷酸化受体群之间

图2 重叠延伸PCR法构建IRF3 gRNA的表达模块注：gRNA表达模块由U6启动子，gRNA序列，gRNA骨架序列三部分组成，在其上下游的酶切位点处设计引物A和D；IRF3 gRNA的位置设计重叠延伸引物B和C

表1 重叠延伸PCR法设计IRF3gRNA的引物序列

引物名称	引物序列	碱基（bp）
引物A	5’-GGGTTCGAATTTATCGATCACGAGACTAGC-3’	30
引物B	5’-GGGCCATTTCTACCAAGGCCGGTGTTTCGTCCTTTCC-3’	37
引物C	5’-GGCCTTGGTAGAAATGGCCCGTTTTAGAGCTAGAAATAGCAAGTTAA-3’	47
引物D	5’-GGGGTACCGAATTCGCTAGCTCTCGA-3’	26

表1 重叠延伸PCR法设计IRF3gRNA的引物序列

引物名称	引物序列	碱基（bp）
引物A	5’-GGGTTCGAATTTATCGATCACGAGACTAGC-3’	30
引物B	5’-GGGCCATTTCTACCAAGGCCGGTGTTTCGTCCTTTCC-3’	37
引物C	5’-GGCCTTGGTAGAAATGGCCCGTTTTAGAGCTAGAAATAGCAAGTTAA-3’	47
引物D	5’-GGGGTACCGAATTCGCTAGCTCTCGA-3’	26

图3 重叠延伸PCR获取IRF3 gRNA表达模块与克隆载体pGM-simple T-IRF3-gRNA的筛选注：3A：重叠延伸PCR扩增生成IRF3 gRNA的表达模块；1.引物A和D对IRF3 gRNA的DNA序列的扩增产生475 bp片段；2.引物A和B对表达载体pLenti-U6-gRNA-PGK-Neo的扩增产生378 bp片段；3.引物C和D对表达载体pLenti-U6-gRNA-PGK-Neo的扩增产生114 bp片段；4. DL1000 DNA Marker。3B：克隆载体的筛选；1~10.通过引物A和D对十个克隆的筛选；11.DL2000 DNA Marker

图4 表达载体的构建与鉴定注：4A：利用ClaI和NheI双酶切pLenti-U6-gRNA-PGK-Neo表达载体，并回收线性化载体；1. pLenti-U6-gRNA-PGK-Neo表达载体经ClaI和NheI双酶切产生7400 bp的线性化载体；2. DL 15000 DNA Marker；3.未酶切的表达载体对照组质粒。4B：利用ClaI和NheI双酶切克隆载体pGM-simple T-IRF3-gRNA，并回收IRF3 gRNA表达模块；1.克隆载体经ClaI和NheI双酶切后产生475 bp的IRF3 gRNA；2. DL 2000 DNA Marker。4C：IRF3 gRNA的Sanger测序结果；灰色阴影区域显示测序结果与IRF3 gRNA的插入序列相一致

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Construction of interferon regulatory factor 3 guided RNA eukaryotic expression vector

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Construction of interferon regulatory factor 3 guided RNA eukaryotic expression vector