| 三种鸭防御素真核表达载体的构建及其表达分析 |
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| 引用本文： | 卢顺,高其双,彭霞,刘武,陈洁,陶弼菲,陈志华,童伟文,周莉,华娟,王连芳.三种鸭防御素真核表达载体的构建及其表达分析[J].湖北农业科学,2016(24):6597-6600. |
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| 作者姓名： | 卢顺 高其双 彭霞 刘武 陈洁 陶弼菲 陈志华 童伟文 周莉 华娟 王连芳 |
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| 作者单位： | 武汉市农业科学技术研究院畜牧兽医科学研究所,武汉,430208 |
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| 基金项目： | 武汉市农业科学技术研究院创新项目(CX201609-09 |
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| 摘 要： | 为研究鸭防御素的真核表达,人工合成了鸭防御素AvBD2、AvBD10和AvBD12基因,并将其插入到真核表达质粒pDoubleEx-EGFP-flag-N中构建重组表达质粒;重组表达质粒经酶切鉴定正确后,分别转染HEK293细胞进行表达,用RT-PCR和Westem-blot鉴定目的基因的表达情况。结果表明,酶切鉴定证实目的基因成功插入到真核表达质粒中;真核表达质粒转染HEK293细胞24 h后,在荧光显微镜下可见绿荧光蛋白表达;RT-PCR能检测到目的基因mRNA,但Western-blot没有检测到目的蛋白的表达条带。
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| 关 键 词： | 鸭防御素 真核表达 HEK293细胞 |
Construction of Eukaryotic Expression Vectors of Three Duck Defensins and Their Expression Analysis |
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| Abstract: | To study the eukaryotic expression of duck defense, duck defensins genes AvBD2, AvBD10 and AvBD12 were ar-tificia11y synthesized and inserted into the eukaryotic expression p1asmid pDoub1eEx-EGFP-f1ag-N to construct recombinant ex-pression p1asmids. After identification by enzyme digestion, recombinant expression p1asmids were transfected into HEK293 ce11s respective1y. And the expression of target genes were ana1yzed by using RT-PCR and Westem-b1ot. The resu1ts showed that target genes were inserted into the eukaryotic expression p1asmid successfu1; 24 hours after the transfection of recombi-nant expression p1asmids into HEK293 ce11s, the expression of green f1uorescent protein were visib1e under the f1uorescence microscope; RT-PCR cou1d detected the target genes' mRNA, but western b1ot did not detect the expression of target pro-teins. This study constructed eukaryotic expression vectors of three duck defenses, and try to express the defensins in HEK293 ce11s. It provided reference for further research of eukaryotic expression of duck defensins. |
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| Keywords: | duck defensins eukaryotic expression HEK293 ce11 |
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