文章摘要
苏建华,李慧敏,缪 珩,鲁一兵,蒋秀芹.介导人血红素加氧酶-1基因真核表达质粒的构建及鉴定[J].南京医科大学学报,2008,28(1):47~50
介导人血红素加氧酶-1基因真核表达质粒的构建及鉴定
Construction and verification of eukaryotic expression plasmid carring human HO-1 gene
投稿时间:2007-04-29  
DOI:10.7655
中文关键词: 血红素加氧酶-1  真核表达质粒  基因重组
英文关键词: heme oxygenase-1  eukaryotic expression plasmid  gene recombination
基金项目: 江苏省卫生厅科研发展重点课题(H200514)
作者单位
苏建华 南京医科大学第二附属医院内分泌科,江苏 南京 210011 
李慧敏 南京医科大学第二附属医院内分泌科,江苏 南京 210011 
缪 珩 南京医科大学第二附属医院内分泌科,江苏 南京 210011 
鲁一兵 南京医科大学第二附属医院内分泌科,江苏 南京 210011 
蒋秀芹 南京医科大学第二附属医院内分泌科,江苏 南京 210011 
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中文摘要:
      目的:构建含人血红素加氧酶-1(HO-1)基因的重组pcDNA3.1质粒并进行鉴定?方法:利用分子生物学技术,从人肝脏标本中提取出RNA,进行RT-PCR后,做TA克隆,成功获得pMD/19-HO-1质粒,采用限制性内切酶BamHⅠ?EcoRⅠ从pMD/19-HO-1质粒中将目的基因片段切出,克隆到质粒pcDNA3.1的相应酶切位点中,构建重组质粒pcDNA3.1-HO-1,采用限制性酶切?DNA测序鉴定,将构建好的质粒瞬时转染ECV304细胞株,Western blot测定HO-1表达?结果:酶切?测序鉴定证实插入位点及方向与预期完全一致,测序证实hHO-1与Gene Bank提供的原始序列完全一致,瞬时转染细胞后HO-1蛋白表达明显增高?结论:成功构建了含人血红素加氧酶-1基因的真核表达质粒,为进一步研究该基因在糖尿病血管并发症中的作用奠定基础?
英文摘要:
      Objective:To construct and certificate eukaryotic expression plasmid carrying human HO-1 gene. Methods:By molecular biological technique,total RNA was extracted from human liver. After RT-PCR and TA cloning,the plasmid of pMD/19-HO-1 was successfully constructed. Then the plasmid was digested with restriction enzymes BamHⅠand EcoRⅠ. Then the human HO-1 gene fragment was cloned into plasmid pcDNA3.1 to construct the recombinant pcDNA3.1-HO-1 plasmid. pcDNA3.1-HO-1 was verified by restriction digestion and gene sequencing,The constructed plasmid were transfected into ECV304 cell line and certificated by Western blot analysis. Results:Identification of pcDNA3.1-HO-1 by restriction digestion and sequencing showed that the target gene was inserted into pcDNA3.1 correctly; the sequence of the target gene by DNA sequencing was identical with that of NCBI gene bank; the plasmid could increase the expression of human HO-1 obviously. Conclusion:pcDNA3.1-HO-1 was constructed successfully,which will help to study the effect of human HO-1 on vasculopathy in diabetes mellitus.
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