文章摘要
郭 泽,龙卫国,曹伯良,张爱霞.HDGF真核表达质粒的构建?表达及其生物活性检测[J].南京医科大学学报,2010,(5):602~606
HDGF真核表达质粒的构建?表达及其生物活性检测
Construction and expression of pEGFP-HDGF and detection of its bioactivity
投稿时间:2009-11-04  
DOI:10.7655
中文关键词: 肝癌衍生生长因子  绿色荧光蛋白  U87细胞株
英文关键词: HDGF  GFP  U87 cells
基金项目:南京医科大学科技发展基金重点项目资助(07NMUZ005);横向合作项目(Van Andel Institute)(ky1011711084000092)
作者单位
郭 泽 南京医科大学病理学系,卫生部抗体技术重点实验室,江苏 南京 210029 
龙卫国  
曹伯良  
张爱霞  
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中文摘要:
      目的:构建pEGFP-HDGF 真核表达质粒,转入胶质瘤U87细胞中,对其表达及生物学活性进行检测?方法:PCR扩增HDGF编码区序列,构建pEGFP-HDGF真核表达质粒,脂质体转染U87细胞,G418筛选稳定表达单克隆,荧光显微镜观察绿色荧光,RT-PCR及Western blot检测融合蛋白在转染细胞中的整合及表达?通过U87细胞增殖实验对HDGF-GFP融合蛋白进行初步功能鉴定?结果:pEGFP-HDGF表达质粒转染U87细胞后,荧光显微镜下可见绿色荧光,RT-PCR?Western blot结果证实在U87细胞中有HDGF-GFP融合蛋白的表达?细胞增殖实验的结果显示,稳定表达pEGFP-HDGF的U87细胞生长速率明显高于对照空载组细胞(P=0.006)?结论:pEGFP-HDGF真核表达质粒构建成功,且HDGF对于胶质瘤U87细胞株有着生长刺激作用,这将有助于胶质瘤发生发展的分子机制研究?
英文摘要:
      Objective:To construct and identify pEGFP-HDGF over expression plasmid in U87 cells. Methods:HDGF fragment was amplified by RT-PCR method and then inserted into pEGFP-N1 vector. The level of HDGF was identified by RT-PCR and western blot after transfection of positive recombinant plasmid into glioma cell line U87. The biological activity of HDGF-GFP fusion protein was tested by the proliferation assay. Results:Fluorescence microscopy demonstrated that pEGFP-HDGF was successfully transfected into U87 cells. Over expression of HDGF was confirmed by RT-PCR and western blot. The proliferation rate of U87 cell transfected with pEGFP-HDGF plasmid was significantly higher than that of negative control. Conclusion:pEGFP-HDGF over expression plasmid with biological activity was successfully constructed and stablely transfected into U87 cells.
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