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

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。

Abstract:

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.