人酸性调宁蛋白calponin-3与EGFP的融合表达及其细胞定位  

Expression and Subcellular Localization Analysis of Recombinant Human Calponin-3-EGFP Fusion Protein

作　　者：卢春花[1] 张昭颖[1] 李游 白如梦 Lu Chunhua;Zhang Zhaoying;Li You;Bai Rumeng(College of Life Science and Technology, Guangxi University, Nanning, 53000)

机构地区：[1]广西大学生命科学与技术学院

出　　处：《基因组学与应用生物学》2018年第7期3214-3220,共7页Genomics and Applied Biology

基　　金：国家自然科学基金资助项目(81360324);“广东省普通高校功能蛋白质研究暨南大学重点实验室”开放课题(2012001);广西教育厅科研项目(2013YB005);2016年度广西高等教育创优计划教学相关项目-优势特色专业项目(优质本科专业);广西大学博士启动基金(XBZ120159)共同资助

摘　　要：调宁蛋白家族进化保守,广泛分布于肌细胞与非肌细胞。目前发现该蛋白家族有碱性、中性和酸性3个异构体,它们在非肌细胞中的功能尚不明确,而酸性调宁蛋白calponin-3是被研究最少的家族成员。本研究通过RT-PCR的方法从人支气管上皮细胞HBE中克隆编码calponin-3蛋白的CNN3基因,克隆至真核表达载体pEGFP-N1。重组质粒pEGFP-N1-CNN3经酶切鉴定和DNA测序正确后,用于瞬时转染细胞。Western blotting检测293T,A549和SMMC-7721细胞内重组绿色荧光融合calponin-3蛋白的瞬时表达,激光共聚焦显微镜分析calponin-3-绿色荧光融合蛋白与罗丹明-鬼笔环肽标记F-actin的细胞定位。结果表明成功构建了真核表达载体pEGFP-N1-CNN3并在293T、A549和SMMC-7721细胞中高效表达;通过绿色荧光结合免疫荧光染色确定calponin-3与F-actin共定位。成功构建calponin-3蛋白的表达载体,在细胞中瞬时表达并检测其细胞亚定位,为进一步研究calponin-3蛋白的生物学功能奠定基础。The calponins protein family was evolutionary highly conserved and expressed in both smooth muscle and non-muscle cells. To date, three isoforms of calponin （basic, neutral and acidic calponin） were found in the vertebrates and their precise function in non-muscle cells is unclear. Moreover, the acidic calponin （calponin-3） is the far least studied family member. In this current study, DNA fragments encoding caiponin-3 （CNN3） was obtained by RT-PCR from human bronchial epithelial （HBE） cells and inserted into eukaryotic expression vector pEGFP-N 1. The recombinant plasmid pEGFP-N1-CNN3 was confirmed by DNA sequencing and enzyme digestion analysis and then was transiently transfected into cells. Transient expression of EGFP-fused calponin-3 in 293T, A549 and SMMC-7721 cells was verified by Western blotting analysis. Intracellular localization of EGFP fluorescent and rhodamine phalloidin-labeled F-actin was detected by confocal laser scanning microscopy. Results showed that recombinant expression vector pEGFP-N 1-CNN3 was constructed and effectively expressed in 293T, A549 and SMMC-7721 cells. Furthermore, EGFP fluorescent combined with immunofluorescent staining revealed co-localization ofcalponin-3 and F-actin in these cells. The successful vector construction, transient expression, and subcellular localization detection ofcalponin-3 would provide basis for its further biological function study.

关 键 词：Calponin-3 绿色荧光蛋白 表达 细胞定位 

分 类 号：R346[医药卫生—基础医学]