白喉毒素受体真核表达载体的构建和鉴定.doc


白喉毒素受体真核表达载体的构建和鉴定#
潘晨琛,冉艳丽,巨富荣,张胜祥**
(兰州大学生命科学学院,兰州 730000)
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摘要:目的:将白喉毒素受体基因装入 p-EGFP-N1,构建 DTR 的真核表达载体,建立由白
喉毒素介导的条件性细胞敲除系统。方法:以含有 DTR 核酸序列的转基因小鼠基因组为模
版进行 PCR 扩增,电泳回收 DTR 片段,并装入 p-EGFP-N1 质粒构建 DTR 的真核表达载
体。通过磷酸钙法将 p-DTR-EGFP-N1 重组质粒转染到 HepG2 细胞,免疫组化分析 DTR-EGFP
融合蛋白的表达,p-DTR-N1 重组质粒转染 Hela 细胞,经白喉毒素处理,荧光镜下检测荧光
的表达,分析细胞形态。结果:PCR 获得了 DTR 的 CDS 全序列,测序结果与文献报道一
致,结果表明 p-DTR-EGFP-N1 可表达 DTR-EGFP 融合蛋白,但 EGFP 未表现出绿色荧光。
p-DTR-N1 转染后细胞由白喉毒素不敏感转变为敏感,表明白喉毒素受体的真核表达载体已
经成功构建。该系统的构建为细胞条件性敲除的研究奠定了基础。
关键词:动物学;真核表达载体;白喉毒素受体;磷酸钙转染;细胞谱系敲除
中图分类号:Q952
Construction and identification of diphtheria toxin receptor
eukaryotic expression vector
PAN Chenchen, RAN Yanli, JU Furong, ZHANG Shengxiang
(School of Life Sciences, Lanzhou University, LanZhou 730000)
Abstract: [Objective]: To establish an effective conditional cell ablation system, we reconstructed
an eukaryotic vector expressing diphtheria toxin receptor by amplifying and cloning DTR gene
into p-EGFP-N1. [Methods]: The genome of a transgenic mouse containing diphtheria toxin
receptor was amplified by PCR, and DTR gene was retrieved by electrophoresis. The eukaryotic
vector of DTR was constructed by inserting the DTR into p-EGFP-N1. The HepG2 cells were
transfected by calcium phosphate method with the reconstructed plasmid p-DTR-EGFP-N1 and
the fusion protein DTR-EGFP was examined by immuno