张志崇,王春生,张秋婷,朴善花,苗向阳,安铁洙

• 1:东北林业大学生命科学学院
• 2:中国农业科学院北京畜牧兽医研究所

摘要(Abstract)：

为了建立大量获取中国林蛙抗菌肽的方法,根据GenBank中的中国林蛙皮肤抗菌肽Temporin-1CEa基因的mRNA序列(EU624139)设计一对特异性引物,以提取的中国林蛙皮肤总RNA反转录出的cDNA为模板,将扩增的编码序列与pEASY-T3克隆载体连接获得Tem-T3;利用酶切、连接等分子生物学手段,将GFP基因连入Tem-T3克隆载体,再经酶切获得Tem-GFP片段,并插入真核表达载体pcDNA3.1,最终得到Tem-GFP-pcD-NA3.1重组质粒;利用脂质体转染法将该质粒转入绵羊成纤维细胞,48 h后可在荧光倒置显微镜下观察到GFP的绿色荧光表达;qPCR数据分析显示,与对照组相比,转染Tem-GFP-pcDNA3.1的绵羊成纤维细胞中融合蛋白Tem-GFP的表达量可提高约300倍。本研究为构建Temporin-1CEa基因山羊乳腺特异表达载体提供依据。

关键词(KeyWords)： 中国林蛙;抗菌肽;绵羊成纤维细胞;瞬时表达

Abstract:

Keywords:

基金项目(Foundation): 国家转基因生物新品种培育科技重大专项(2009ZX08008-004B);; 国家自然科学基金资助项目(30771538,31000990)

作者(Author): 张志崇,王春生,张秋婷,朴善花,苗向阳,安铁洙

DOI: 10.13326/j.jea.2012.01.001

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