Expression and localization of Copine-3-enhanced green fluorescent protein fusion protein
10.16098/j.issn.0529-1356.2021.03.009
- Author:
You LI
1
;
Li-Ming XU
1
;
Qi-Rain ZHANG
1
;
Chun-Hua LU
1
;
Su-Fang YANG
2
Author Information
1. College of Life Science and Technology, Guangxi University
2. Department of Reproductive Health and Infertility, Guangxi International Zhuang Medical Hospital
- Publication Type:Journal Article
- Keywords:
Bronchial epithelial cell;
Copine-3;
Eukaryotic expression;
Human;
Immunofluorescence;
Western blotting
- From:
Acta Anatomica Sinica
2021;52(3):384-389
- CountryChina
- Language:Chinese
-
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-Nl. The recombinant plasmid pEGFP-Nl-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-Nl 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-Nl-CPNE3 is constructed successfully, and Copine-3 protein is localized in cytoplasm and nucleus.
