小鼠PDZ9的克隆及亚细胞定位分析.pdfVIP

第十一届中南地区实验动物科技交流会论文集 小鼠 PDZ9 的克隆及亚细胞定位分析 14 姚峰 ，刘鑫，王宗保，吴端生，陈春芳，贺震，余坚，谭翔文 （南华大学实验动物学部，衡阳 421001 ） 【摘要】目的克隆小鼠基因 PDZ9 并构建 pEGFP-C1 载体进行亚细胞定位分析。 方法 利用 RT-PCR 技术扩增小鼠基因 PDZ9 开放阅读框序列。将目的基因片段插入 pGEM-T 载 体中, 经双酶切和测序鉴定后, 定向克隆至荧光表达载体pEGFP-C1, EcoRI 及 SalI 双酶切鉴 定。脂质体法转染 COS-7 细胞, 荧光显微镜下观察确定其亚细胞定位。结果 PDZ9 片断长 度为 961bp, 与预期结果一致; 亚细胞定位结果表明, 该蛋白定位于细胞核中。结论 获得了 PDZ9 基因片段，构建 pEGFP-C1- PDZ9 质粒，为下一步功能研究奠定基础。 【关键词】 PDZ9; 克隆; 转染; 亚细胞定位 Cloning and Subcellular Location of the Novel Gene PDZ9 YAO Feng, LIU Xin,WANG Zong-bao , WU Duan-sheng ,CHEN Chun-fang,HE Zheng , YU Jian,TANG Xing-wen (Department of Laboratory Animal Science,University of South China,Hengyang 421001,China) 【Abstract 】 Objective To investigate the cloning, subcellular location of the novel mouse gene PDZ9. Methods PDZ9 gene was amplified by RT-PCR, the PCR product s was cloned into pGEM-T vector. Then it was inserted to plasmid pEGFP-C1 ( enhanced green fluorescent protein) . To identify the subcellular location of PDZ9, the recombinant plasmid was transfected to COS-7 cells. Restricted enzymes analysis and DNA sequencing showed that the sequence of the recombinant plasmid pEGFPC1-PDZ9 was correct. Results The results of PCR and sequence presented that the sequence of the PDZ9 gene was a 961bp, Green fluorescence signal was distributed uniformly in cytoplasm and cell membrane. Conclusions The recombinant prokaryotic expression vector and the PDZ9 obtained successfully in this study would be benefited for further investigations on the function of PDZ9 gene. PDZ源于三个蛋白质的首字母, 分别为哺乳动物突触后密度蛋白PSD295( postsynaptic density protein)、