Construction and expressions of recombinant plasmid encoding human aquaglyceroporin 9

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.