Copine-3-增强型绿色荧光蛋白融合蛋白在细胞中的表达和定位  

Expression and localization of Copine-3-enhanced green fluorescent protein fusion protein

作　　者：李游 徐丽明 张琦敏 杨素芳 卢春花[1] LI You;XU Li-ming;ZHANG Qi-min;YANG Su-fang;LU Chun-hua(College of Life Science and Technology,Guangxi University,Nanning 530004,China;Department of Reproductive Health and Infertility,Guangxi International Zhuang Medical Hospital,Nanning 530201,China)

机构地区：[1]广西大学生命科学与技术学院,南宁530004 [2]广西国际壮医医院生殖健康与不孕症科,南宁530201

出　　处：《解剖学报》2021年第3期384-390,共7页Acta Anatomica Sinica

基　　金：国家自然科学基金(81360324,81760500);广西省自然科学基金(2017GXNSFAA198362,2018GXNSFDA050009);广西省研究生教育创新计划项目(YCSW2018045)。

摘　　要：目的构建增强型绿色荧光蛋白(p EGFP)-N1-CPNE3真核表达载体,检测Copine-3蛋白在细胞中的表达和定位。方法利用RT-PCR从人支气管上皮细胞(HBE)中扩增得到编码Copine-3蛋白的CPNE3基因,并亚克隆至p EGFP-N1真核表达载体。将酶切鉴定和测序正确的pEGFP-N1-CPNE3重组质粒转染293T和H1299细胞。激光扫描共焦检测Copine-3-EGFP融合蛋白的细胞定位,Western blotting检测亚细胞组分中Copine-3蛋白的表达,免疫组织化学检测肺腺癌患者癌临床样品中的Copine-3蛋白定位。结果编码Copine-3蛋白的CPNE3基因克隆到了p EGFP-N1真核表达载体中,并在293T和H1299细胞中表达。通过免疫荧光染色、Western blotting和免疫组织化学确定Copine-3蛋白定位于细胞质和细胞核。结论成功构建p EGFP-N1-CPNE3真核表达载体,Copine-3蛋白定位于细胞质和细胞核。Objective To construct the eukaryotic expression vector plasmid enhanced green fluorescent protein(p EGFP)-N1-CPNE3,and identify the expression and localization of Copine-3 protein in cells.Methods The Copine-3 coding sequences(CPNE3)was amplified by RT-PCR from human bronchial epithelial(HBE)cells and inserted into eukaryotic expression vector pEGFP-N1.The recombinant plasmid p EGFP-N1-CPNE3 was confirmed by endonuclease digestion and sequencing before it was transfected into 293 T and H1299 cells.Cellular localization of Copine-3-EGFP fusion protein was detected by con-focal laser scanning.Expression of Copine-3 in 293 T and H1299 cells was detected by Western blotting analysis.Localization of Copine-3 in clinical samples of the lung adenocarcinoma patients was detected by immunohistochemistry.Results CPNE3 was successfully constructed into the eukaryotic expression vector p EGFP-N1 and expressed in 293 T and H1299 cells.Furthermore,the location of Copine-3 protein in cytoplasm and nucleus was determined by immunofluorescence staining,immuno Western blotting and immunohistochemistry in those cells and clinical samples.Conclusion The eukaryotic expression vector p EGFP-N1-CPNE3 is constructed successfully,and Copine-3 protein is localized in cytoplasm and nucleus.

关 键 词：Copine-3 真核表达 支气管上皮细胞 免疫荧光 免疫印迹法 人 

分 类 号：R34[医药卫生—基础医学]