SHISAL1基因真核表达质粒构建及其在肝癌细胞中表达
Construction of Eukaryotic Expressional Plasmid of SHISAL1 Gene and Its Expression in Hepatocellular Car-cinoma Cells
DOI: 10.12677/BIPHY.2022.103005, PDF,    国家自然科学基金支持
作者: 吴文昊, 周莉莉, 石 静, 严 辉, 陈 帅, 孙达权*：贵州医科大学基础医学院，贵州 贵阳
关键词: SHISAL1基因；基因克隆；真核表达；肝癌细胞； SHISAL1 Gene； Gene Cloning； Eukaryotic Expression； Hepatocellular Carcinoma Cell
摘要: 本文的目的是克隆人SHISAL1基因和构建其真核表达载体，并在肝细胞癌细胞中表达和分析。以人肝细胞THLE-3总RNA为模板，用RT-PCR获得SHISAL1基因蛋白编码区，通过基因克隆和重组获得真核表达载体pDsRed1-SHISAL1；用脂质体介导法将pDsRed1-SHISAL1导入肝癌细胞HuH-7中，用荧光显微镜和激光共聚焦扫描显微镜观察细胞内红色荧光，用蛋白免疫印迹和免疫荧光染色分析细胞内FZD3的表达情况。结果显示成功克隆人SHISAL1基因蛋白编码区并获得了真核表达质粒pDsRed1-SHISAL1；荧光显微术和蛋白免疫印迹结果显示外源SHISAL1融合蛋白，即SHISAL1-RFP蛋白，在肝癌细胞HuH-7中表达；激光共聚焦扫描显微术和蛋白免疫印迹结果显示SHISAL1可以抑制肝癌细胞HuH-7内源性FZD3表达。本研究成功克隆了SHISAL1基因并在肝癌细胞中表达，并证明SHISAL1影响肝癌细胞内源性FZD3表达。
Abstract: The aims are to clone human SHISAL1 gene and con-struct its eukaryotic expressional vector, and then to express and analyze the protein in hepatocel-lular carcinoma cells. Using THLE-3 total RNA as a template, the protein coding region of SHISAL1 gene was obtained by RT-PCR, and the eukaryotic expressional vector pDsRed1-SHISAL1 was ob-tained by gene cloning and recombination. The recombinant plasmid was introduced into hepato-cellular carcinoma cell line HuH-7 by liposome method. The intracellular red fluorescence was ob-served by fluorescence microscopy and laser confocal scanning microscopy, and the expression of endogenous FZD3 was analyzed by western blot and immunofluorescence staining. The results showed that the protein coding region of human SHISAL1 gene was cloned successfully and the eu-karyotic expression plasmid pDsRed1-SHISAL1 was obtained. Fluorescence microscopy and western blot showed that SHISAL1 fusion protein, namely SHISAL1-RFP fusion protein, was expressed in HuH-7 cells. Laser confocal scanning microscopy and western blot showed that overexpression of SHISAL1 could inhibit the endogenous expression of FZD3 in HuH-7 cells. Collectively, SHISAL1 gene was successfully cloned and expressed in hepatocellular carcinoma cells, and then it was proved that SHISAL1 could affect the expression of endogenous FZD3 in hepatocellular carcinoma cells.
文章引用：吴文昊, 周莉莉, 石静, 严辉, 陈帅, 孙达权. SHISAL1基因真核表达质粒构建及其在肝癌细胞中表达[J]. 生物物理学, 2022, 10(3): 39-45. https://doi.org/10.12677/BIPHY.2022.103005

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