中国医科大学学报

中国医科大学学报

中国医科大学学报 ›› 2009, Vol. 38 ›› Issue (11): 816–.

• 基础医学 • 上一篇    下一篇

hLMO1编码基因重组质粒的构建及蛋白的表达和定位

蔡鑫泽; 顾卉; 刘彤; 李丰   

  1. 中国医科大学附属第一医院中心实验室; 中国医科大学基础医学院细胞生物学教研室; 卫生部细胞生物学重点实验室
  • 出版日期:2009-12-20 发布日期:2010-04-14
  • 通讯作者: 李丰 E-mail:lifengphd15@hotmail.com

Construction of the Recombinant Plasmid of Human LMO1 Gene and the Expression and Localization of Fusion Protein

  • Online:2009-12-20 Published:2010-04-14

摘要:

目的构建hLMO1真核表达载体并证实融合蛋白在细胞内的表达及定位。方法以人胎脑文库cDNA为模板,PCR扩增hLMO1基因cDNA全长,亚克隆至pEGFP表达载体中。将构建的重组质粒进行酶切测序鉴定,并转染到人上皮细胞——HEK293细胞中,提取细胞蛋白进行Western
      blot检测。利用激光扫描共聚焦显微镜观察pEGFP-hLMO1在HEK293细胞内的定位。结果hLMO1基因cDNA全长克隆到了真核表达载体pEGFP中,酶切鉴定片段为471bp。Western
      blot检测到了融合蛋白表达,分子量约为45kDa。pEGFR-LMO1在人HEK293细胞核与细胞质均有表达。结论成功构建了hLMO1基因cDNA全长的真核表达载体,pEGFP-LMO1蛋白定位于HEK293细胞的细胞质和细胞核内。

关键词: hLMO1基因, 质粒构建, 基因表达与定位

Abstract:

Objective To construct the expression plasmid of human LIM domain
      only 1(hLMO1)gene and identify the expression and localization of its
      fusion protein.Methods The hLMO1 coding sequence was amplified by
      polymerase chain reaction by using cDNA library derived from human fetal
      brain as the template and subcloned into pEGFR vector.After the target
      region was identified by enzyme digestion and sequencing,the plasmid was
      transfected into HEK293 cells.The expression of the recombinant plasmid in
      HEK293 cells was detected by Western blot.The localization of pEGFP-LMO1
      in HEK293 cells was observed with laser scanning confocal
      microscopy.Results hLMO1 was constructed into the expressing vector pEGFP
      successfully.The length of the fragment identified by restriction enzyme
      digestion was 471 bp.The expression of pEGFP-LMO1 fusion protein with a
      molecular weight of 45 kDa was detected by Western blot.The pEGFP-LMO1
      fusion protein was localized in both the nucleus and cytoplasm of HEK293
      cells.Conclusion The recombinant plasmid of hLMO1 gene was
      successfully cloned into eukaryotic expressing vector,and the pEGFP-LMO1
      fusion protein was expressed in the nucleus and cytoplasm.

Key words: LIM domain only 1 gene, plasmid construction, gene expression and localization

[1] 张殿宝 ,赵峰 ,张涛 ,林学文 ,王瑞 ,施萍 ,庞希宁 ,. 靶向 REST 基因 TALEN 质粒的快速构建与活性检测[J]. 中国医科大学学报, 2016, 45(10): 883-886.
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