猪肌肉素基因的cDNA克隆与表达.PDF

生 物 工 程 学 报 Chin J Biotech 2008, July 25; 24(7): 1248-1252 Chinese Journal of Biotechnology ISSN 1000-3061 cjb@ © 2008 Institute of Microbiology, CAS CSM, All rights reserved 研究报告 猪肌肉素基因的cDNA 克隆与表达 王伟杰, 李宏基, 韩立强, 王月影, 王静, 李卫华, 林茂旺, 台玉磊, 张志强, 藏猛, 王艳龄, 杨国宇 河南农业大学动物生理生化实验室, 郑州 450002 摘 要: 从人肌肉素基因出发, 在 dbEST 数据库中进行同源性搜索, 找到七个有较高同源性的 Expressed Sequence Tag(DY426490, CF787546, AJ660979, AJ664670, AJ663820, AJ680159, DN106254) 。通过拼接和进一步RT-PCR 实验验证, 获得猪肌肉素基因全长cDNA 序列, 其全长651 bp, 开放阅读框为54~452 bp, 编码有 132 个氨基酸。同源性分析结果 表明, 与人、小鼠和大鼠的肌肉素基因cDNA 编码区(CDS) 同源性分别为87.2%、77.6%和77.9% 。利用克隆出的猪肌肉 素 cDNA, 构建表达载体pGEX-4T-1-musclin, 并在BL21 大肠杆菌中成功表达和纯化了分子量为38.59 kD 的融合蛋白 GST-Musclin, 并运用蛋白印迹技术进行鉴定。 关键词: 猪, 肌肉素基因, 表达, 纯化, 鉴定 Cloning and Expression of the Pig Skeletal Muscle Musclin Gene Weijie Wang, Hongji Li, Liqiang Han, Yueying Wang, Jing Wang, Weihua Li, Maowang Lin, Yulei Tai, Zhiqiang Zhang, Meng Zang, Yanling Wang, and Guoyu Yang Laboratory of Animal Physiology Biochemistry , He’nan Agricultural University, Zhengzhou 450002, China Abstract: We found seven tag sequence with high homology in dbEST by using human musclin gene, and got its cDNA sequence, which consists of 651bp and the open reading frame was 54~452 bp detected by RT-PCR, encoding 132 amino acid residue protein . The new gene has high homology with that of human, mouse and rat, the rate is 87.2%, 77.6% and 77.9%, respectively; the gene fragment was cloned into expression vector pGEX-4T-1, and the recombinant was transformed into E. coli BL21. Induced by IPTG, the fusion protein GST-musclin, a 38.59 kD protein was successfully expressed in E. coli BL21 and identified