人水甘油通道蛋白9重组质粒的构建及表达  

Construction and expressions of recombinant plasmid encoding human aquaglyceroporin 9

作　　者：王川[1] 谭翠[1] 姜政[1] 王丕龙[1] 

机构地区：[1]重庆医科大学附属第一医院消化内科,重庆400016

出　　处：《重庆医科大学学报》2013年第1期20-23,共4页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:81070318);重庆市卫生局科研资助项目(编号:渝卫科教2010-2-100)

摘　　要：目的:构建人水甘油通道蛋白9(aquaglyceroporin9,AQP9)重组质粒,并检测其融合蛋白在真核细胞COS-7中的表达,为阐明AQP9在非酒精性脂肪肝病(nonalcoholic fatty liver disease,NAFLD)中的作用奠定基础。方法:通过RT-PCR方法从人肝脏组织中获得AQP9基因,插入pEGFP-N1质粒,构建重组质粒pEGFP-N1-AQP9,转染COS-7细胞,通过荧光显微镜检测其转染情况,RT-PCR和Western blot检测AQP9在真核细胞中的表达。结果:PCR及酶切鉴定显示插入人AQP9大小正确。测序鉴定显示,1个碱基发生突变,45位:A→G,为无义突变。通过RT-PCR和Western blot检测到AQP9表达,说明AQP9基因在COS-7细胞中表达并与绿色荧光蛋白融合,且具有免疫原性。结论:成功构建了pEGFP-N1-AQP9重组质粒,并在COS-7细胞中表达,为进一步研究AQP9在NAFLD发生发展中的作用奠定基础。Objective：To construct human aquaglyceroporin 9（AQP9）recombinant plasmid and to detect its expressions in COS-7 cells in order to elucidate the role of AQP9 in pathogenesis of nonalcoholic fatty liver disease（NAFLD）.Methods：AQP9 gene was obtained by RT-PCR from human hepatic tissue and was cloned into pEGFP-N1 plasmid.pEGFP-N1-AQP9 recombinant plasmid was constructed,identificated and transfected into COS-7 cells.Transfection was detected by fluorescence microscopy;expressions of AQP9 were detected by RT-PCR and Western blot.Results：PCR and enzyme digestion analysis showed that AQP9 size was right.Equencing analysis found a nonsense mutation,45 th：A→G.RT-PCR and Western blot demonstrated that AQP9 was expressed in COS7 cells,suggesting that pEGFP-N1-AQP9 was expressed in COS-7 cells and had immunogenicity.Conclusions：pEGFP-N1-AQP9 recombinant plasmid is constructed and expressed successfully in COS-7 cells,which would lay a foundation for further study of AQP9 in the pathogenesis of NAFLD.

关 键 词：水甘油通道蛋白9 非酒精性脂肪肝病 重组质粒 

分 类 号：R575.1[医药卫生—消化系统]