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

李游; 徐丽明; 张琦敏; 杨素芳; 卢春花

doi:10.16098/j.issn.0529-1356.2021.03.009

PDF(11725 KB)

解剖学报 ›› 2021, Vol. 52 ›› Issue (3) : 384-390. DOI: 10.16098/j.issn.0529-1356.2021.03.009

细胞和分子生物学

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

李游¹徐丽明¹ 张琦敏¹ 杨素芳^2*卢春花1^*

作者信息 +

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

LI You¹ XU Li-ming¹ ZHANG Qi-min¹ YANG Su-fang^2* LU Chun-hua^1*

Author information +

文章历史 +

摘要

目的构建增强型绿色荧光蛋白（pEGFP)-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pEGFP-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pEGFP-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pEGFP-N1-CPNE3真核表达载体，Copine-3蛋白定位于细胞质和细胞核。

Abstract

Objective To construct the eukaryotic expression vector plasmid enhanced green fluorescent protein(pEGFP)-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 pEGFP-N1-CPNE3 was confirmed by endonuclease digestion and sequencing before it was transfected into 293T and H1299 cells. Cellular localization of Copine-3-EGFP fusion protein was detected by con-focal laser scanning. Expression of Copine-3 in 293T 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 pEGFP-N1 and expressed in 293T 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 pEGFP-N1-CPNE3 is constructed successfully, and Copine-3 protein is localized in cytoplasm and nucleus.

导出引用

李游徐丽明张琦敏杨素芳卢春花. 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[J]. 解剖学报. 2021, 52(3): 384-390 https://doi.org/10.16098/j.issn.0529-1356.2021.03.009

LI You XU Li-ming ZHANG Qi-min YANG Su-fang LU Chun-hua. Expression and localization of Copine-3-enhanced green fluorescent protein fusion protein[J]. Acta Anatomica Sinica. 2021, 52(3): 384-390 https://doi.org/10.16098/j.issn.0529-1356.2021.03.009

中图分类号： R34

参考文献

［1］ Creutz CE, Tomsig JL, Snyder SL, et al. The copines, a novel class of C2 domain-containing, calcium-dependent, phospholipid-binding proteins conserved from Paramecium to humans ［J］. J Biol Chem, 1998, 273(3): 1393-1402.

［2］ Wu YR, Wang XW, Jin YB, et al. Effect of CPNE5 on transcriptional activity of NF-κB ［J］. Bulletin of the Academy of Military Medical Science, 2009, 33(3): 237-240. (in Chinese)

吴彦瑞, 王晓文, 靳雁斌, 等. CPNE5对NF-κB转录调控活性的影响［J］. 军事医学科学院院刊, 2009, 33(3):237-240.

［3］ Yoo JC, Park N, Choi HY, et al. JAB1 regulates CPNE1-related differentiation via direct binding to CPNE1 in HiB5 hippocampal progenitor cells ［J］. Biochem Biophys Res Commun, 2018, 497(1): 424-429.

［4］ Tomsig JL, Sohma H, Creutz CE. Calcium-dependent regulation of tumour necrosis factor-alpha receptor signalling by copine ［J］. Biochem J, 2004, 378(Pt 3): 1089-1094.

［5］ Ramsey CS, Yeung F, Stoddard PB, et al. Copine-Ⅰ represses NF-kappaB transcription by endoproteolysis of p65 ［J］. Oncogene, 2008, 27(25): 3516-3526.

［6］ Zhu B, Zha J, Long Y, et al. Increased expression of copine Ⅵ in patients with refractory epilepsy and a rat model ［J］. J Neurol Sci, 2016, 360: 30-36.

［7］ Oh HJ, Choung HW, Lee HK, et al. CPNE7, a preameloblast-derived factor, regulates odontoblastic differentiation of mesenchymal stem cells ［J］. Biomaterials, 2015, 37: 208-217.

［8］ Park SJ, Lee HK, Seo YM, et al. Dentin sialophosphoprotein expression in enamel is regulated by Copine-7, a preameloblast-derived factor ［J］. Arch Oral Biol, 2018, 86: 131-137.

［9］ Ramsey H, Zhang DE, Richkind K, et al. Fusion of AML1/Runx1 to copine Ⅷ, a novel member of the copine family, in an aggressive acute myelogenous leukemia with t(12;21) translocation ［J］. Leukemia, 2003, 17(8): 1665-1666.

［10］ Tomsig JL, Creutz CE. Copines: a ubiquitous family of Ca(2+)-dependent phospholipid-binding proteins ［J］. Cell Mol Life Sci, 2002, 59(9): 1467-1477.

［11］ Caudell EG, Caudell JJ, Tang CH, et al. Characterization of human copine Ⅲ as a Phosphoprotein with associated kinase activity ［J］. Biochemistry, 2000, 42(39): 13034-13043.

［12］ Fu L, Fu H, Qiao J, et al. High expression of CPNE3 predicts adverse prognosis in acute myeloid leukemia ［J］. Cancer Sci, 2017, 108(9): 1850-1857.

［13］ Tan B, Liu L, Yang Y, et al. Low CPNE3 expression is associated with risk of acute myocardial infarction: a feasible genetic marker of acute myocardial infarction in patients with stable coronary artery disease ［J］. Cardiol J, 2019, 26(2): 186-193.

［14］ Heinrich C, Keller C, Boulay A, et al. Copine-Ⅲ interacts with ErbB2 and promotes tumor cell migration ［J］. Oncogene, 2010, 29(11): 1598-1610.

［15］ Lin HC, Zhang FL, Geng Q, et al. Quantitative proteomic analysis identifies CPNE3 as a novel metastasis-promoting gene in NSCLC ［J］. J Proteome Res, 2013, 12(7): 3423-3433.

［16］ Lin H, Zhang X, Liao L, et al. CPNE3 promotes migration and invasion in nonsmall cell lung cancer by interacting with RACK1 via FAK signaling activation ［J］. J Cancer, 2018, 9(22): 4215-4222.

［17］ Chen Z, Jiang Z, Zhang W, et al. Silencing the expression of copine-Ⅲ enhances the sensitivity of hepatocellular carcinoma cells to the molecular targeted agent sorafenib ［J］. Cancer Management Res, 2018, 10: 3057-3067.

［18］ Hao HX, Wang FL, Ai GQ, et al. Expression of RTCB in different tissues of mice［J］. Acta Anatomica Sinica, 2019, 50(3): 346-350. (in Chinese)

郝海霞, 王富龙, 艾桂青, 等. RTCB在小鼠不同组织中的表达［J］. 解剖学报, 2019, 50(3): 346-50.

［19］ Zhu ZL, Tan J, Deng H. Nucleus translocation of membrane/cytoplasm proteins in tumor cells ［J］. Journal of Zhejiang University(Medical Science), 2019, 48(3): 318325. (in Chinese)

朱紫菱, 谈静, 邓红. 肿瘤细胞膜/质蛋白转位入核研究进展［J］. 浙江大学学报（医学版）, 2019, 48(3): 318-325.