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

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.