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  

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

作　　者：许会静 刘晴 闫冰 刘微 张磊 郭健 姜琳 李淼[2] 孙美艳 李艳 XU Huijing;LIU Qing;YAN Bing;LIU Wei;ZHANG Lei;GUO Jian;JIANG Lin;LI miao;SUN Meiyan;LI Yan(Medical Laboratory,Jilin Medical College,Jilin 132013,China;Department of Neurology,China-Japan Union Hospital,Jilin University,Changchun 130033,China;Center of Antibody,Jilin Medical College,Jilin132013,China)

机构地区：[1]吉林医药学院检验学院实验中心,吉林吉林132013 [2]吉林大学中日联谊医院神经外科,吉林长春130033 [3]吉林医药学院抗体中心,吉林吉林132013

出　　处：《吉林大学学报（医学版）》2019年第6期1193-1198,I0015,共7页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金面上项目资助课题(81771304);国家自然科学基金青年项目资助课题(81601234,81601073);吉林省财政厅省直卫生专项项目资助课题(SCZSY201616);吉林省中医药管理局科研项目资助课题(2019140);吉林省吉林市科技创新发展计划项目资助课题(201750246);吉林省大学生创新创业项目资助课题(201837)

摘　　要：目的:构建水通道蛋白4(AQP4)真核表达载体pmCherry-N1-hAQP4-M23,检测AQP4-M23蛋白在中国仓鼠肺细胞系V79中的表达。方法:采用PCR方法扩增hAQP4-M23基因,经双酶切后与真核表达载体pmCherry-N1连接,构建pmCherry-N1-hAQP4-M23重组表达质粒。采用脂质体将其转染至V79细胞中,将细胞随机分为对照组(未转染重组质粒的V79细胞)和转染组(转染重组质粒的V79细胞)。利用RT-PCR法和荧光显微镜检测2组细胞中hAQP4-M23基因的表达;取视神经脊髓炎(NMO)患者血清中抗AQP4抗体,与2组细胞结合,采用免疫荧光和水通透性法检测2组细胞中hAQP4-M23的活性。结果:成功构建AQP4真核表达载体;荧光显微镜观察,转染组细胞膜清晰表达红色荧光;免疫荧光检测,转染组细胞膜呈现绿色荧光,表明转染组细胞膜AQP4-M23蛋白可与NMO患者血清成分结合;与对照组比较,转染组细胞中hAQP4-M23活性升高(P<0.05)。结论:成功构建表达AQP4基因的真核表达载体,并在V79细胞系中成功表达hAQP4-M23蛋白。Objective:To construct the eukaryotic expression vector pmCherry-N1-hAQP4-M23 of 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.

关 键 词：视神经脊髓炎 水通道蛋白4 真核表达载体 基因转染 仓鼠 

分 类 号：Q71[生物学—分子生物学]