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

doi:10.16098/j.issn.0529-1356.2021.03.009

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

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^*

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

收稿日期:2020-02-20 修回日期:2020-03-30 出版日期:2021-06-06 发布日期:2021-06-06
通讯作者: 杨素芳;卢春花 E-mail:chlu@gxu.edu.cn
基金资助:
国家自然科学基金;国家自然科学基金;广西自然科学基金;广西自然科学基金;广西研究生教育创新计划项目

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*

1. College of Life Science and Technology, Guangxi University, Nanning 530004, China; 2.Department of Reproductive Health and Infertility, Guangxi International Zhuang Medical Hospital, Nanning 530201, China

Received:2020-02-20 Revised:2020-03-30 Online:2021-06-06 Published:2021-06-06
Contact: YANG Su-fang;LU Chun-hua E-mail:chlu@gxu.edu.cn

摘要/Abstract

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。

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

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.

Key words: Copine-3, Eukaryotic expression, Bronchial epithelial cell, Immunofluorescence, Western blotting, Human

中图分类号:

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.

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.

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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

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