靶向酪氨酸蛋白激酶EphA7基因siRNA表达载体的构建及筛选.docVIP

  靶向酪氨酸蛋白激酶 EphA7 基因 siRNA 表 达载体的构建及筛选# 张水军，张弓，李捷，赵永福，郭文治* 5 10 15 20 25 30 35 40 45 （郑州大学第一附属医院肝胆胰外科，郑州 450052） 摘要：目的：构建针对酪氨酸蛋白激酶 EphA7 基因的小干扰 RNA(small interference RNA, siRNA)表达载体，以用于后续的 RNA 干扰研究。方法：根据 Genbank 上人类 EphA7 基因 的核苷酸序列及 siRNA 的设计原则，选择 3 段序列：638-656nt、713-731nt 和 1456-1474nt 作为干扰片段，人工合成针对 EphA7 的发夹样结构 siRNA 互补双链寡核苷酸。经过退火、 质粒线性化以及体外重组等过程，构建抑制 EphA7 基因表达的干扰载体 pRNA-siEphA7-1、 2 和 3。将干扰载体转染入感受态大肠杆菌 DH5α后进行克隆和筛选，PCR 鉴定及基因测序 验证干扰载体的准确性。结果：合成的正义及反义寡核苷酸，经退火处理、DNA 片段纯化 及回收，电泳可见 118bp 附近的清晰条带。以空载体 pRNA-U6.1/Neo 为对照，PCR 扩增筛 选鉴定，得到 310bp 左右的扩增片段的阳性克隆，证明已经插入 pRNA-U6.1/Neo 载体。对 3 个重组质粒进行测序，结果证实插入的寡核苷酸序列正确。结论：成功构建了针对 EphA7 基因表达的干扰载体 pRNA-siEphA7-1、2 和 3，为以后的研究奠定基础。 关键词：EphA7；siRNA；载体；筛选 中图分类号：R735.7 Construction and identification of siRNA expression vector targeting at EphA7 ZHANG Shuijun, ZHANG Gong, LI Jie, ZHAO Yongfu, GUO Wenzhi (Department of Hepatobiliary and Pancreatic Surgery, the First Affiliated Hospital of Zhengzhou University, ZhengZhou 450052) Abstract: Objective To construct the eukaryotic expression vector pRNA-siEphA7 for the tyrosine kinase receptor EphA7 gene. Methods According to the nucleotide sequence of the human EphA7 gene in Genbank and the principles of siRNA design, 3 segment sequences were chosen: 638-656nt, 713-731nt and 1456-1474nt. All of the three sequences were combined with the corresponding plasmid vector pRNA-u6.1/Neo restriction recognition sites and transcription terminator at the end of them. After annealing, endonuclease reaction and reconstitution in vivo, the siRNA expressing vectors pRNA-siEphA7 targeting EphA7 gene was constructed. The three vectors were transfected into E. coli DH5α. After cloning and selection, PCR and gene sequencing analysis was used to verify the accuracy of recombinant vector. Results The sense and antisense oligonucleotide can form the double-stranded after annealing process. RT-PCR showed that a clear band around 118bp. By using the empty vector pRNA-u6.1/Neo as control, we finally found the ampl