人FUT3基因真核表达载体的构建与表达

Construction of human FUT3 eukaryotic expression vector and its expression in human breast adenocarcinoma cells

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载体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的表达.结果:成功获得人全长FUT3基因并构建了真核表达载体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.

AIM： To construct the human FUT3 （α 1, 3-fucosyltransferase） eukaryotic expression vector and analyze its expression in human breast adenocarcinoma MDA-MB-231 cells. METHODS： The full-length FUT3 cDNA was obtained by reverse transcription-polymerase chain reaction （RT-PCR） and cloned into pMD18-T simple vector for sequence analysis. Then the FUT3 gene was subcloned into pEGFP-C1 plasmid. The resulting recombinant vector pEGFP-C1-FUT3 was identified by digestion with restriction endonucleases and transfected into MDA-MB-231 cells. A stably transfected cell line was established using G418 selection. The expression of FUT3 wasobserved under a fluorescence microscope and examined by semi-quantitative RT-PCR. RESULTS： The full-length human FUT3 cDNA was successfully obtained, and the recombinant plasmid pEGFP-C1-FUT3 was successfully constructed. After transfection into MDA-MB-231 cells, green fluorescence （green fluorescent protein） was observed. Semi-quantitative RTPCR analysis showed that FUT3 was highly expressed in MDA-MB-231 cells. CONCLUSION： The FUT3 eukaryotic expression vector pEGFP-C1-FUT3 that can express FUT3 in MDA-MB-231 cells is constructed successfully and can be used to study the biological functions of the FUT3 gene.