Eukaryotic expression of human NOVA1 protein and identification of its anti-hypoxia activity.
- Author:
Hualing LI
1
;
Bei LV
1
;
Ling KONG
1
;
Xinhong CHEN
1
;
Sujuan ZHU
2
Author Information
- Publication Type:Journal Article
- Keywords: anti-hypoxia; distribution; eukaryotic expression vector; human NOVA1 protein; transfection
- MeSH: Animals; Blotting, Western; Cell Hypoxia; Genetic Vectors; Humans; PC12 Cells; Plasmids; RNA, Messenger; RNA-Binding Proteins; genetics; metabolism; Rats; Transfection
- From: Chinese Journal of Biotechnology 2016;32(4):507-517
- CountryChina
- Language:Chinese
- Abstract: The aim of this study was to construct the eukaryotic expression vector of pCMV-Myc-NOVA1 based on NOVA1 gene, and to screen the optimum expression condition after transfecting to PC12 cells, and further to explore the distribution of NOVA1 protein in PC12 cells using cell immunohistochemistry, and to identifyits anti-hypoxia activity. According to the NOVA1 gene sequence of NCBI database, we designed the upstream and downstream primers, and performed polymerase chain reaction (PCR) to amplify the full length cDNA coding sequence using pCR4-TOPO-NOVA1 as a template. The products were digested by restriction endonuclease SalⅠand XhoⅠ, and conjugated to the eukaryotic expression vector ofpCMV-Myc followed by validating by digestion and direct sequencing. Subsequently, the validated pCMV-Myc-NOVA1 was transfected to PC12 cells followed by optimizing of transfection ratio and transfection time, and identified by qPCR, Western blotting and cell immunohistochemistry respectively. After validation by digestion and direct sequencing, the eukaryotic expression vector of pCMV-Myc-NOVA1 was correctly constructed. The optimum transfection ratio of plasmid to Lipo 2000 was 1:2.5, and the optimum transfection time was 72 h. At the optimum transfection condition, the expression level of NOVA1 mRNA and protein significantly increased, and after transfection of pCMV-Myc-NOVA1, NOVA1 protein mainly distributed in cell nucleus and cytoplasm. After 6 h hypoxia, the cell proliferation activity was significantly increased compared to that of the control and pCMV-Myc group. Our findings provided a reference for exploring the mechanism of NOVA1, and also a technical support for potential drug development of NOVA1.
