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Construction of human FUT3 eukaryotic expression vector and its expression in human breast adenocarcinoma cells
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- Author:
- No author available
- Journal Title:
- WORLD CHINESE JOURNAL OF DIGESTOLOGY
- Issue:
- 31
- DOI:
- 10.3969/j.issn.1009-3079.2009.31.005
- Key Word:
- α 1;3-岩藻糖基转移酶Ⅲ;增强型绿色荧光蛋白;基因克隆
Abstract： 目的:构建增强型绿色荧光蛋白为报告基因的pEGFP-C1-FUT3真核表达载体,分析其在细胞系MDA-MB-231中的表达.方法:采用RT-PCR扩增FUT3全长基因片段,克隆至pMD18-T载体进行测序分析,将FUT3亚克隆至pEGFP-C1表达载体并酶切鉴定.利用脂质体将重组真核表达栽体pEGFP-C1-FUT3转染入人乳腺癌细胞MDA-MB-231中,经G418筛选获得稳定转染的细胞系,荧光显微镜观察及RT-PCR检测FUT3的表选.结果:成功获得人全长FUT3基因并构建了真核表达栽体pEGFP-C1-FUT3,体外转染MDA-MB-231细胞后荧光显微镜下可见绿色荧光蛋白的表达,半定量RT-PCR检出高水平表达的FUT3.结论:成功构建了增强型绿色荧光蛋白为报告基因的FUT3真核表达载体,并在MDA-MB-231中稳定表达,为进一步研究FUT3的生物学功能提供基础.
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