引用本文:王 川,谭 翠,姜 政,王丕龙.人水甘油通道蛋白9重组质粒的构建及表达[J].重庆医科大学学报,2013,(1):20~23
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
DOI:
中文关键词:  水甘油通道蛋白9  非酒精性脂肪肝病  重组质粒
英文关键词:aquaglyceroporin 9  nonalcoholic fatty liver disease  recombinant plasmid
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作者单位
王 川,谭 翠,姜 政,王丕龙 重庆医科大学附属第一医院消化内科重庆 400016 
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中文摘要:
      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。
英文摘要:
      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 ob-tained 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. E-quencing analysis found a nonsense mutation,45 th:A→G. RT-PCR and Western blot demonstrated that AQP9 was expressed in COS-7 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.
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