AQP4真核表达载体的构建和AQP4-M23蛋白在中国仓鼠肺细胞系V79中的表达

doi:10.13481/j.1671-587x.20190601

Abstract

Abstract: Objective: To construct the eukaryotic expression vector pmCherry-N1-hAQP4-M23of aquaporin 4 (AQP4),and to detect the expression of hAQP4-M23 protein in the Chinese hamster lung cell line V79. Methods: The hAQP4-M23 gene was amplified by PCR method,and ligated into the pmCherry-N1 after double digestion to construct the recombinant plasmid pmCherry-N1-hAQP4-M23. The plasmids were transfented into V79 cells by lipidosome,and the cells were randomly divided into control group (the V79 cells without recombinant plasmid transfection) and transfection group (the V79 cells with recombinant plasmid transfection).The expressions of hAQP4-M23 in the cells in two groups were detected by RT-PCR method and fluorescence microscope;the AQP4 antibody in serum of the neuromyelitis optica(NMO) patient was bound to the cells in two groups and the activities of hAQP4-M23 in the cells in two groups were detected by immunofluorescence and water permeability assay. Results: The sequencing results showed that the AQP4 eukaryotic expression vector was successfully constructed.The fluorescence microscope results showed that the cell membrane in transfection group clearly expressed the red fluorescence.The immunofluorescence detection results showed that the cell membrane in transfection group presented the green fluorescence,indicating that the AQP4-M23 protein of cell membrane in transfection group could be bound to the serum components of the NMO patients.Compared with control group,the activity of hAQP4-M23 in the cells in transfection group was increased significantly (P<0.05). Conclusion: The eukaryotic expression vector expressing AQP4 gene is successfully constructed and the AQP4-M23 protein is successfully expressed in the V79 cell line.

XU Huijing, LIU Qing, YAN Bing, LIU Wei, ZHANG Lei, GUO Jian, JIANG Lin, LI miao, SUN Meiyan, LI Yan. Construction of eukaryotic expression vector of AQP4 and expression of AQP4-M23 protein in Chinese hamster lung cell line V79[J].Journal of Jilin University(Medicine Edition), 2019, 45(06): 1193-1198.

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Construction of eukaryotic expression vector of AQP4 and expression of AQP4-M23 protein in Chinese hamster lung cell line V79

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