脆性X精神发育迟缓基因1(FMR1)真核表达载体的构建及其应用  

Construction and application of eukaryotic expression vector of fragile X mental retardation 1(FMR1) gene

作　　者：杨文静[1] 富显果[1] 廖娟[1] 郭小艳[1] 兰风华[1] 

机构地区：[1]厦门大学附属东方医院全军计划生育优生优育技术研究所医学遗传研究室,福建福州350000

出　　处：《细胞与分子免疫学杂志》2016年第11期1513-1516,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：福建省重点科技项目资助(2010Y0045)

摘　　要：目的构建人脆性X精神发育迟缓基因1(FMR1)真核表达载体,建立稳定转染FMR1的He La细胞系。方法采用PCR扩增出人FMR1的c DNA编码区序列,利用DNA重组技术将其定向插入p EGFP-N2真核表达载体中,并对重组质粒进行双酶切及DNA测序鉴定。用脂质体将验证的重组质粒转染至He La细胞,通过G418筛选建立FMR1稳定转染的He La细胞系。进一步运用Western blot法、免疫荧光染色结合激光扫描共聚焦显微镜技术鉴定FMR蛋白(FMRP)在He La细胞中的表达及定位。结果双酶切和DNA测序结果表明p EGFP-N2-FMR1真核表达质粒构建成功。Western blot和激光共聚焦显微镜结果显示GFP-FMRP融合蛋白在He La细胞中成功表达且主要定位在细胞质。结论成功建立FMR1稳定转染的He La细胞系并可以表达FMRP。Objective To construct a eukaryotic expression vector of human fragile X mental retardation 1（FMR1） gene and establish stably transfected He La cells.Methods The full-length FMR1 c DNA fragment was amplified by PCR and inserted into eukaryotic expression vector p EGFP-N2 using restriction enzyme.The recombinant plasmid p EGFP-N2-FMR1,after identified by restriction digestion and DNA sequencing,was transfected into He La cells by lipofectamine 2000.The stably transfected cell line was obtained by screening with G418.The expression and subcellular distribution of FMR protein was identified by Western blotting and immunofluorescence staining combined with laser-scanning confocal microscopy.Results Restriction digestion and DNA sequencing revealed that the eukaryotic expression plasmid of p EGFP-N2-FMR1 was successfully constructed.Besides,Western blotting and immunofluorescence staining showed that GFP-FMR protein was expressed in He La cells,which mainly was localized in the cytoplasm.Conclusion The recombinant eukaryotic expression vector of p EGFP-N2-FMR1 has been constructed successfully and stably expressed FMR protein in He La cells.

关 键 词：脆性X精神发育迟缓基因1(FMR1) 真核表达 He La细胞 免疫荧光技术 

分 类 号：R392-33[医药卫生—免疫学] Q786[医药卫生—基础医学]