| 人ST3Gal Ⅰ基因真核表达载体的构建与表达 |
| 投稿时间：2010-05-20 点此下载全文 |
| 引用本文：崔红霞,岳丽玲,刘吉成.人ST3Gal Ⅰ基因真核表达载体的构建与表达[J].医学研究杂志,2010,39(10):30-33 |
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| 基金项目:国家自然科学基金资助项目（30772751） |
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| 中文摘要:目的构建增强型绿色荧光蛋白为报告基因的pEGFP-N1-ST3Gal Ⅰ真核表达载体，分析其在人乳腺癌细胞系MCF-7中的表达，筛选稳转细胞株，为进一步研究乳腺癌细胞膜连α2,3唾液酸与转移潜能调控提供细胞模型。方法采用RT-PCR扩增ST3Gal Ⅰ全长基因片段，克隆至pEGFP-N1载体进行测序分析，用脂质体将重组真核表达载体pEGFP-N1-ST3Gal Ⅰ转染人乳腺癌细胞MCF-7中，经荧光显微镜观察及RT-PCR检测hST3Gal I的表达，G418抗性筛选稳转细胞株。结果成功构建了真核表达载体pEGFP-N1- ST3Gal Ⅰ，体外转染MCF-7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hST3Gal Ⅰ，获得稳转细胞株。结论成功构建了增强型绿色荧光蛋白为报告基因的hST3Gal I真核表达载体，并在MCF-7中稳定表达, 为进一步研究乳腺癌细胞膜连α2,3唾液酸水平增高与其侵袭和转移潜能的调控提供实验基础。 |
| 中文关键词:α2,3唾液酸转移酶 Ⅰ增强型绿色荧光蛋白 转染 表达 |
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| Construction and Expression of hST3Gal Ⅰ Eukaryotic Expression Vector in Human Breast Carcinoma Cells |
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| Abstract:ObjectiveTo construct the human ST3Gal Ⅰ (α 2, 3 sialyltransferase) eukaryotic expression vector and analyze its expression in MCF-7 cells.MethodsThe human ST3Gal Ⅰ cDNA was obtained and amplificated by reverse transcription-polymerase chain reaction (RT-PCR). Then the hST3Gal Ⅰ gene was incorporation into pEGFP-N1 plasmid. The recombinant vector pEGFP-N1-ST3Gal Ⅰ was identified and transfected into MCF-7 cells. A stably transfected cell line was established using G418 resistance selection.The expression of hST3Gal Ⅰ was observed under a fluorescence microscope and examined by semi-quantitative RT-PCR. ResultsThe recombinant plasmid pEGFP-N1-ST3Gal Ⅰ was successfully constructed.After it being transfected into MCF-7 cells, green fluorescence could bee observed.Results of semi-quantitative RT-PCR analysis displayed that the level of hST3Gal Ⅰ mRNA was significantly increased in MCF-7 cells.ConclusionThe hST3Gal Ⅰ eukaryotic expression vector pEGFP-N1-ST3Gal Ⅰ that can express hST3Gal Ⅰ in MCF-7 cells is constructed successfully and can be used to study the correlation of breast cancer metastatic potential with the overpression of hST3Gal Ⅰ gene. |
| keywords:α2, 3 sialyltransferase I Enhanced green fluorescent protein Transfection Expression |
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