【摘要】 [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。更多还原

【Abstract】 [Objective]To construct XRCC1 siRNA eukaryotic expression vector,and thus lay a basis for establishing XRCC1-defecient cell line and the further study on the function of XRCC1.[Methods]Hairpin siRNA Retroviral Vector for XRCC1 was constructed according to cDNA sequence of XRCC1 gene provided by GenBank;By means of gene engineering,the siRNA Eukaryotic GFP Expression Vector for XRCC1 was constructed through inserting the aim fragment from gel extraction(including siRNA and U6 promotor)into pEGFP-C1 vector.The recombinant pEGFP-C1 vector was identified by restriction endonuclease and DNA sequence.[Results]Hairpin siRNA Retroviral Vector for XRCC1 and the siRNA Eukaryotic GFP Expression Vector for XRCC1 were constructed successfully.[Conclusion]The siRNA Karyoatic Expression Vector for XRCC1 was constructed successfully and laid a basis for the establishment of XRCC1-deficient cell strain.更多还原