文章摘要
张大为,朱 进,陈仁杰,冯振卿.EB病毒潜伏膜蛋白1胞外区融合蛋白的表达及纯化[J].南京医科大学学报,2008,28(4):420~423
EB病毒潜伏膜蛋白1胞外区融合蛋白的表达及纯化
Expression and purification of GST-LMP-1 extracellular domain fusion protein of EB virus
投稿时间:2007-10-20  
DOI:10.7655
中文关键词: 鼻咽癌  EB病毒  LMP1  胞外区  重组基因
英文关键词: nasopharyngeal carcinoma  epstein-barr virus  latent membrane protein 1,extracellular peptide  recombinant gene
基金项目:南京医科大学科技发展基金重点项目(2005NYD2D13);南京军区“122”工程资助项目
作者单位
张大为 南京医科大学第二附属医院耳鼻咽喉科江苏 南京 210029 
朱 进 南京军区军事医学研究所江苏 南京 210002 
陈仁杰 南京医科大学第二附属医院耳鼻咽喉科江苏 南京 210029 
冯振卿 南京医科大学卫生部抗体技术重点实验室江苏 南京 210029 
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中文摘要:
      目的:在大肠杆菌中表达EB病毒潜伏膜蛋白1(LMP1)胞外肽段与谷胱甘肽S-转移酶(GST)融合蛋白,并用该融合表达蛋白检测EB 病毒相关鼻咽癌(NPC) 患者血清中的特异性抗体。方法:用BamHⅠ和EcoRⅠ将含LMP1 胞外区重组基因的质粒pMD18-LMP1双酶切后,克隆到pGEX-4T-2中。重组质粒经酶切鉴定、核酸序列分析后转化大肠杆菌BL21(DE3),以异丙基硫代-β-D-半乳糖苷(IPTG) 诱导表达融合蛋白,表达的蛋白经谷胱甘肽S-转移酶柱亲和层析纯化,纯化的蛋白经Western blot 法检测鉴定。结果:重组表达质粒经酶切鉴定为阳性,核酸序列分析正确。SDS-PAGE 和Western blot 结果显示表达的重组蛋白分子质量约为32.2 ku,此抗原能够与鼻咽癌患者血清中抗体特异性结合。结论:成功获得了LMP1 胞外区重组基因表达的蛋白,并证明原核表达的LMP1胞外肽段保持了原有的抗原性,能够与EBV相关鼻咽癌患者血清中的抗体特异性结合。
英文摘要:
      Objective:To express structural GST-LMP-1 fusion protein in E.coli,detecting and analyzing its antibodies in NPC patients’ sera of EBV associated malignancies with Western-blot. Methods:The plasmid pMD18-LMP1 containing the recombinant extracellular peptide gene of LMP1 of EBV was digested with BamHⅠ and EcoRⅠ,and cloned into expression vector pGEX-4T-2. The constructed vector which had been identified by enzyme digestion and nucleotide sequence analysis was transformed into BL21(DE3). After induced with IPTG,the recombinant protein was purified with GST affinity chromatography,and confirmed by Western blot. Results:The recombinant plasmid was constructed correctly. SDS-PAGE analysis showed that the recombinant protein was about 32.2 ku. The purified protein could be used as an antigen to detect specific antibodies in the sera of patients with NPC by Western blot. Conclusion:The recombinant extracellular peptide gene of LMP1 of EBV could be expressed with high performance,and the antigenicity of the purified protein was certificated in sera of patients with NPC, which was associated with EBV.
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