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[目的]构建XRCC1基因的发卡样siRNA(small interfere RNA)真核表达载体,为建立XRCC1缺陷的细胞株,及进一步研究XRCC1的功能奠定基础。[方法]根据GenBank提供的XRCC1基因cDNA序列及软件构建发卡样XRCC1基因siRNA逆转录病毒载体;选择酶切位点,将包括U6启动子和siRNA序列的片段切下,并亚克隆至入CMV启动子指导的pEGFP-C1荧光蛋白表达载体,构建XRCC1基因siRNA绿色荧光蛋白真核表达载体,构建载体通过荧光测序鉴定。[结果]通过分子克隆和琼脂糖凝胶电泳鉴定,成功构建了发卡样XRCC1基因siRNA逆转录病毒载体;经过亚克隆和琼脂糖凝胶电泳及测序鉴定成功获得pEGFP-C1-XRCC1 siRNA真核表达载体。[结论]成功构建pEGFP-C1-XRCC1 siRNA真核表达载体,为建立XRCC1蛋白缺陷细胞株提供材料。
[Objective] To construct a eukaryotic expression vector for small RNA of XRCC1 gene and lay the foundation for establishing a cell line with XRCC1 deficiency and further studying the function of XRCC1. [Method] The hairpin-like XRCC1 gene siRNA retroviral vector was constructed based on the cDNA sequence of XRCC1 gene provided by GenBank and software. The restriction site was selected and the fragment including U6 promoter and siRNA sequence was excised and subcloned into CMV promoter Subclone pEGFP-C1 fluorescent protein expression vector to construct XRCC1 gene siRNA green fluorescent protein eukaryotic expression vector, construction vector identified by fluorescent sequencing. [Result] The hairpin-like XRCC1 gene siRNA retroviral vector was successfully constructed by molecular cloning and agarose gel electrophoresis. The eukaryotic expression of pEGFP-C1-XRCC1 siRNA was successfully obtained by subcloning and agarose gel electrophoresis and sequencing Vector. [Conclusion] The eukaryotic expression vector pEGFP-C1-XRCC1 siRNA was successfully constructed and provided material for the establishment of XRCC1 protein deficient cell line.