Construction of eukaryotic expression of mouse derived pcDNA3. 1 ⁃3 × Flag⁃c⁃NUP85 plasmid and its partial function research
10.19405/j.cnki.issn1000-1492.2023.05.015
- Author:
Yan Yao
1
;
Shuxian Wang
1
;
Yincui Wu
1
;
Shuang Hu
1
;
Ying Hu
1
;
Linxin Pan
2
;
Tao Xu
1
Author Information
1. School ofPharmacy,Anhui Medical University,Hefei 230032
2. School ofLife Science ,Anhui Medical University,Hefei 230032
- Publication Type:Journal Article
- Keywords:
NUP85;
RAW264. 7 cells;
proliferation;
apoptosis;
TNF⁃α;
IL⁃6
- From:
Acta Universitatis Medicinalis Anhui
2023;58(5):794-799
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct a mouse derived pcDNA3. 1 ⁃3 × Flag⁃c⁃NUP85 expression plasmid and observe its effect on expression of inflammation factors in LPS⁃induced RAW264. 7 cells , as well as on the proliferation and apoptosis of RAW264. 7 cells.
Methods:The NUP85 gene was amplified by PCR to construct pcDNA3. 1 ⁃3 × Flag-c⁃NUP85 eukaryotic expression plasmid. The pcDNA3. 1 ⁃3 × Flag⁃c vector was divided with enzymes. The purified PCR product was ligated with the vector, and the ligated product was transformed into bacterial competent cells. After identification by enzyme digestion , sequencing and analysis were performed. Then , it was transfected into RAW264. 7 cells , and the blank plasmid without NUP85 gene was set as the control group. The effect on cell proliferation and apoptosis were detected by CCK⁃8 assay and flow cytometry , and the expression of inflammatory cytokines such as tumor necrosis factor⁃α (TNF⁃α ) and interleukin⁃6 (IL⁃6) in LPS⁃induced RAW264. 7 cells was detected by Western blot and ELISA.
Results:Enzyme digestion identification and Western blot results showed that pcDNA3. 1 ⁃3 × Flag⁃c⁃NUP85 eukaryotic expression plasmid was successfully constructed and expressed. The results of CCK⁃8 assay showed that the cell survival rate of NUP85 overexpression group was significantly lower than that of control group after 24 h[(0. 55 ± 0. 03) vs (0. 67 ± 0. 05) , F = 30. 98 , P < 0. 05 ] . The results of flow cytometry showed that the cell apoptosis rate of NUP85 overexpression group was higher than that of control group[( 15. 78 ±1. 05)% vs ( 13. 40 ± 0. 47)% , F = 75. 38 , P < 0. 05] . The results of Western blot and ELISA showed that after transfection of pcDNA3. 1 ⁃3 × Flag⁃c⁃NUP85 , the expression of TNF⁃α and IL⁃6 in RAW264. 7 cells were higher than those in the control group ,with statistical significance (P < 0. 05) .
Conclusion :NUP85 can inhibit the proliferation and promote apoptosis in LPS⁃stimulated RAW264. 7 cells , and NUP85 can promote the expression of inflammatory cytokines IL⁃6 and TNF⁃α in LPS⁃stimulated RAW264. 7 cells.
- Full text:2024081210562099491鼠源pcDNA3.1-3×...质粒的构建及其部分功能研究_姚燕.pdf
