MyD88基因真核表达载体的构建及表达

Construction of eukaryotic expression vector of MyD88 gene and detection of its expression

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.

AIM ： To construct eukaryotic expression vector of the gene of myeloid differentiation factor 88 （MyD88） and to detect its expression in GES-1 cells. METHODS： Total RNA was extracted from the mononuclear ceils of human peripheral blood. The full length cDNA of MyD88 gene was amplified by RT- PCR and then cloned into pcDNA3.1 /myc-His （ - ） A vector by Kpn I and Nhe I restriction enzyme sites. The gene was identified and confirmed by sequencing and then was transfected into GES-I cells. The expressed MyD88 protein was detected by Western blot analysis. RESULTS： The sequencing results confirmed that the eukaryotic expression vector of MyD88 was correctly constructed and the results of Western blot showed its good expression in GES-1 cells. CONCLUSION： pcDNA3. 1/myc-His （ - ） A-MyD88 has been successfully constructed and expressed in GES-1 cells, which provides a foundation for further investigation of the molecular constitution and function of MyD88.