文章摘要
苏 毅,何雅丽,王子露,李建民,徐 艳.pEGFP-N1/hIL-1ra重组真核表达载体的构建及其在人牙龈成纤维细胞中的瞬时表达[J].南京医科大学学报,2008,28(12):1533~
pEGFP-N1/hIL-1ra重组真核表达载体的构建及其在人牙龈成纤维细胞中的瞬时表达
Construction of the eukaryote expression vector carrying encoding gene of human interleukin-1 receptor antagonist and its transient expression in human gingival fibroblast cells
投稿时间:2008-07-03  
DOI:10.7655
中文关键词: 重组人白细胞介素1受体拮抗剂  真核表达载体  瞬时转染  人牙龈成纤维细胞
英文关键词: rhIL-1ra  eukaryote expression vector  transient transfection  human gingival fibroblast cells
基金项目:江苏省自然科学基金资助项目(BK2003422)
作者单位
苏 毅 南京医科大学口腔医学研究所, 江苏 南京 210029 
何雅丽 南京医科大学口腔医学研究所, 江苏 南京 210029 
王子露 南京医科大学口腔医学研究所, 江苏 南京 210029 
李建民 南京医科大学基础医学院,江苏 南京 210029 
徐 艳 南京医科大学口腔医学研究所, 江苏 南京 210029 
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中文摘要:
      目的:构建重组人白细胞介素1受体拮抗剂(rhIL-1ra)基因真核表达载体pEGFP-N1/hIL-1ra,瞬时转染人牙龈成纤维细胞并检测其表达,为牙周炎症的基因治疗提供实验基础?方法:TRIzol法提取人乳腺癌组织总RNA进行RT-PCR,将纯化的扩增产物hIL-1ra与克隆载体pMD18-T连接?转化,测序正确后将重组体pMD18-T/hIL-1ra与真核表达质粒pEGFP-N1分别进行双酶切,连接后转化感受态细菌E.coli TOP10?将构建正确的pEGFP-N1/hIL-1ra重组质粒DNA瞬时转染人牙龈成纤维细胞,荧光显微镜观察绿色荧光的表达,RT-PCR方法检测其基因的表达?结果:构建的pEGFP-N1/hIL-1ra真核表达载体经PCR及双酶切鉴定均表明人IL-1ra基因已与pEGFP-N1正确重组?瞬时转染人牙龈成纤维细胞后能观察到绿色荧光,RT-PCR可得到目的片段?结论:成功构建了重组质粒pEGFP-N1/hIL-1ra,瞬时转染人牙龈成纤维细胞后检测到其基因水平的表达,为炎性细胞因子拮抗剂基因用于牙周炎抗炎治疗奠定了实验基础?
英文摘要:
      Objective:To construct the recombinant pEGFP-N1/hIL-1ra and test its transient expression in human gingival fibroblast cells, this may be useful of the treatment of periodontitis. Methods:Total RNA was extracted from human mammary cancer tissue. RT-PCR was performed to amplify the hIL-1ra encoding gene. Then PCR product was purified and cloned into pMD18-T. After DNA sequencing,both of the recombinant vector pMD18-T/hIL-1ra and plasmid pEGFP-N1 were digested with HindⅢ and BamHⅠ respectively. IL-1ra gene fragment was ligated into plasmid pEGFP-N1. The recombinant plasmid DNA was transformed into E.coli competent cells TOP 10 and positive clones were selected and tested. After being transfected by pEGFP-N1/hIL-1ra,the transient expression of IL-1ra gene in human gingival fibroblast(HGF) cells were detected by fluorescence microscope and RT-PCR. Results:hIL-1ra encoding gene fragment was 531 bp and was inserted into the eukaryotic expression vector pEGFP-N1/hIL-1ra correctly. And HGF cells,which were identified by both fluorescence microscope and RT-PCR,had a transient expression of IL-1ra after transfection. Conclusion:The new recombinant expression vector pEGFP-N1/hIL-1ra was constructed successfully and the HGF cells which could express IL-1ra transiently were also successfully made.
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