MyD88基因真核表达载体的构建及表达
Journal of the Fourth Military Medical University（2009）
Abstract
目的:构建人髓样分化因子88(MyD88)基因编码区序列(cDNA)的真核表达载体,观察其在GES-1细胞中的表达.方法:从健康人外周血单个核细胞中提取总RNA,应用RT-PCR方法扩增MyD88基因cDNA全长序列,经NheI和KpnI酶切位点,插入到pcDNA3.1/myc-His(-)A质粒中,构建成MyD88基因的真核表达载体;用脂质体Lipo-fectamine2000将其转染入人胃黏膜细胞株GES-1细胞中,Western Blot检测其在GES-1细胞中的表达.结果:重组载体经酶切鉴定和测序证实目的基因正确无误;Western Blot结果显示MyD88基因在GES-1细胞具有良好的表达.结论:成功构建了pcDNA3.1/myc-His(-)A-MyD88真核表达载体,并在GES-1细胞中进行了表达,为进一步研究MyD88的结构和功能奠定了基础.
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Key words
gene transfection,RT-PCR,Western Blot,gene cloning,MyD88
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