人ST3Gal Ⅰ基因真核表达载体的构建与表达

Construction and Expression of hST3Gal Ⅰ Eukaryotic Expression Vector in Human Breast Carcinoma Cells

目的构建增强型绿色荧光蛋白为报告基因的pEGFP-N1-ST3GalⅠ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ST3GalⅠ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-ST3GalⅠ转染人乳腺癌细胞MCF-7中,经荧光显微镜观察及RT-PCR检测hST3GalI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-ST3GalⅠ,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hST3GalⅠ,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hST3GaI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。

Objective To construct the human ST3Gal Ⅰ （α2, 3 sialyhransferase） eukaryotic expression vector and analyze its expression in MCF-7 cells. Methods The human ST3Gal Ⅰ cDNA was obtained and amplifieated 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 transfeeted 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. Results The recombinant plasmid pEGFP - N1 - ST3Gal Ⅰ was successfully constructed. After it being transfeeted into MCF -7 ceils, 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. Conclusion The 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.