文章摘要
沙玉根,黄松明,张爱华,赵 非,陈荣华.荧光表达载体Podocin过表达对HEK293细胞中CD2AP分布的影响[J].南京医科大学学报,2007,(6):527~529533
荧光表达载体Podocin过表达对HEK293细胞中CD2AP分布的影响
Construction of expression vector for podocin and its effect of transfection on CD2AP distribution in HEK293 cells
投稿时间:2006-11-05  
DOI:10.7655
中文关键词: podocin  表达载体  CD2AP
英文关键词: podocin  expression vector  CD2AP
基金项目:国家自然科学基金资助项目(30100081),南京市卫生局资助项目(ZKM06043)
作者单位
沙玉根 南京医科大学附属南京市儿童医院肾脏科江苏 南京 210008 
黄松明 南京医科大学附属南京市儿童医院肾脏科江苏 南京 210008 
张爱华 南京医科大学附属南京市儿童医院肾脏科江苏 南京 210008 
赵 非 南京医科大学附属南京市儿童医院肾脏科江苏 南京 210008 
陈荣华 南京医科大学儿科研究所江苏 南京 210029 
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中文摘要:
      目的:构建podocin荧光表达载体,并观察其转染HEK293后对CD2AP细胞内分布的影响。方法:PCR扩增NPHS2 cDNA全长,通过T/A克隆连接至pGEMT-easy载体,应用XhoⅠ和BamHⅠ双酶切后,再将NPHS2全长亚克隆入pEGFP-C2。将构建好的荧光表达载体pEGFP-NPHS2转染HEK293细胞,通过免疫荧光的方法观察转染前后CD2AP细胞内的分布情况。结果:①成功构建pEGFP-NPHS2荧光表达载体;②Podocin表达载体转染HEK293细胞后,CD2AP由核周转为弥散的细胞质分布。结论:Podocin转染HEK293细胞可使CD2AP由核周转为弥散的细胞质分布。
英文摘要:
      Objective:To construct the podocin immunofluorence expression vector and observe its effects of transfection on CD2AP distribution in HEK293 cells. Methods:The pGEMT-easy vector containing the full-length cDNA encoding the human podocin was cloned and digested with BamHI and XhoI. The digested full-length podocin was subcloned into pEGFP-C2. The constructed plasmids were transfected into HEK293 cells,and its effect on CD2AP distribution were observed by immunofluorescence. The interaction between podocin and CD2AP was detected by immunoprecipitation. Results:①The pEGFP-NPHS2 expression vector was successfully constructed and podocin exclusively located on HEK293 cell membrane.② After podocin transfection, CD2AP redistributed from perinucleus to cytoplasm in HEK293. Conclusion:Podocin can recruit CD2AP to redistribute from perinucleus to cytoplasm in HEK293 cells.
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