Construction of eukaryotic expression vector for rat Akt1 gene and its expression in 293 cells
10.3969/j.issn.1673-8225.2009.15.041
- VernacularTitle:大鼠Akt1基因真核表达载体构建及其在293细胞中的表达
- Author:
Rui ZHANG
;
Chun SONG
;
Hui WANG
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2009;13(15):2991-2994
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: Akt gene plays an important role in cell survival, proliferation, metabolism, and apoptosis. Although mechanism of Akt gene still remains unclear, it, a major element of survival signal, has aroused much attention in the domain of molecular biological research. OBJECTIVE: To construct eukaryotic expression vector pDC316-Akt1 for Aktl gene and to observe the expression in 293 cells. DESIGN, TIME AND SETTING: An observational study was performed at Molecular Biological Laboratory of Academy of Military Medical Sciences of Chinese PLA between September and December 2006. MATERIALS: pCD316 plasmid was provided by Benyuan Zhengyang Company; DH5α and 293 cells were reserved in our laboratory. METHODS: Aktl DNA segment was cloned from total RNA of hepatic tissue using RT-PCR and inserted into eukaryotic expressing vector pDC316 between EcoRI and Hind Ⅲ sites after sequencing. And then, 293 cells were transfected with the recombinant by liposome. MAIN OUTCOME MEASURES: Aktl gene content in transfected 293 cells using Westem blotting. RESULTS: Sequencing test indicated that Aktl amino acid sequence was in accordance with wild Aktl sequence. Akt-pDC316 was transcripted in 293 cells, and Akt1 protein with relative molecular weight of 55 000 was highly expressed in 293 cells when assayed using Western blotting. CONCLUSION: Eukaryotic expression vector for Aktl gene highly expresses in 293 cells.
