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山东大学学报(医学版)

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人IL-26的基因克隆及其真核表达载体的构建

刘义庆1,2,王来城2,焦玉莲2,张捷2,马春燕2,崔彬2,高新谱2,刘正敏2,张雪2,赵跃然1,2   

  1. 1. 山东省医学科学院基础医学研究所, 山东 济南 250062;2. 山东大学山东省立医院科研中心, 山东 济南 250021
  • 收稿日期:2006-03-29 修回日期:1900-01-01 出版日期:2006-06-24 发布日期:2006-06-24
  • 通讯作者: 刘义庆

〖KH+9.2mmD]Cloning of human interleukin26 and constructing ofits eukaryotic expression vector

LIU Yiqing1,2,WANG Lai-cheng2,JIAO Yu-lian2,ZHANG Jie2,MA Chunyan2,CUI Bin2,GAO Xinpu2,LIU Zheng-in2,HANG Xue2,ZHAO Yue-ran1,2   

  1. 1.Institute of Basic Medicine, Shandong Academy of Medical Sciences, Jinan 250062, Shandong, China;2. Medical Research Center, Shandong Provincial Hospital, Shandong University, Jinan 250021, Shandong, China
  • Received:2006-03-29 Revised:1900-01-01 Online:2006-06-24 Published:2006-06-24
  • Contact: LIU Qing-yi

摘要: 基因,构建其真核表达载体并探讨在真核细胞中的表达。方法:提取人外周血单个核细胞总RNA,逆转录聚合酶链反应(RTPCR)扩增hIL26基因;目的片段与pMD18T载体连接,转化大肠杆菌E.coliDH5α,菌落PCR、酶切分析和DNA序列分析鉴定重组克隆。用SalⅠ和EcoRⅠ将目的片段切下,插入pIRES2EGFP的相应位点,构建表达载体pIRES2EGFP/hIL26并进行酶切鉴定。将重组质粒用非脂质体试剂Fugene 6转染至COS7细胞中。结果:重组克隆载体pMD18T/hIL26内插入片段序列与GenBank上报道的hIL26基因序列完全一致;pIRES2EGFP/hIL26经酶切鉴定与预期结果一致;荧光显微镜观察可见转染后的细胞有荧光出现,Western blotting 鉴定证明hIL26基因得到了有效表达。结论:成功构建了hIL26基因的真核表达载体并在真核细胞中获得了有效表达。

Abstract: To clone the gene of human interleukin26(hIL26)and to construct its eukaryotic expression vector. Methods: Total RNA was extracted from the human peripheral blood monocytes,and reverse transcriptasepolymerase chain reaction(RTPCR)was performed. The PCR product was inserted into cloning vector pMD18T to construct the recombinant plasmid of hIL26pMD18T/hIL26. Recombinants were transformed into E.coliDH5α and screened with colony PCR and restriction analysis. The gene of hIL26 cloned was sequenced. The hIL26 gene fragment obtained from pMD18T/hIL26 was digested with SalⅠ and EcoRⅠ, and then inserted into pIRES2EGFP that was cut with XhoⅠ and EcoRⅠ. Recombinants were identified by colony PCR and restriction analysis. The recombinant expression plasmid pIRES2EGFPhIL26 was

Key words: Interleukin26, Gene cloning, COS7 cells, Eukaryotic expression vector

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