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
沈瑞明 1凌彩虹 1黄俏佳1
作者信息
- 1. 福建医科大学福总临床医学院,福建,福州,350025
- 折叠
摘要
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将其转染人人胃黏膜细胞株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/mye-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.
Abstract
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 cells of human peripheral blood. The full length cDNA of MyD88 gene was amplified by RTPCR 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-1 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.
关键词
人髓样分化因子88/逆转录-PCR/基因克隆/基因转染/蛋白质印迹技术Key words
MyD88/RT-PCR/gene cloning/gene transfection/Western Blot引用本文复制引用
基金项目
南京军区医药卫生科研基金(08MA100)
南京军区福州总医院留学归国人员专项基金(2004037)
出版年
2009
NETL
NSTL
维普
万方数据