Construction of SOCS3 recombinant adenovirus and its expression in porcine primary adipocytes.
- Author:
Shuangjuan YANG
1
;
Chengquan XU
;
Jiangwei WU
;
Gongshe YANG
Author Information
1. Laboratory of Animal Fat Deposition and Muscle Development, Northwest A & F University, Yangling 712100, China.
- Publication Type:Journal Article
- MeSH:
Adenoviridae;
genetics;
metabolism;
Adipocytes;
metabolism;
Animals;
Cloning, Molecular;
Green Fluorescent Proteins;
biosynthesis;
genetics;
HEK293 Cells;
Humans;
RNA, Messenger;
biosynthesis;
genetics;
Recombinant Fusion Proteins;
biosynthesis;
genetics;
Suppressor of Cytokine Signaling 3 Protein;
Suppressor of Cytokine Signaling Proteins;
biosynthesis;
genetics;
Swine;
Transfection
- From:
Chinese Journal of Biotechnology
2010;26(4):462-469
- CountryChina
- Language:Chinese
-
Abstract:
In order to construct recombinant adenovirus vector expressing Suppressor of cytokine signaling 3 (SOCS3) and obtain infectious adenoviral particles, SOCS3 gene was amplified from plasmid pcDNA3-SOCS3 and subcloned into the adenovirus shuttle plasmid pAdTrack-CMV. After sequence confirmation, the recombinant shuttle plasmid pAdTrack-CMV-SOCS3 was linearized by Pme I, and then transformed into BJ5183 competent cell, the recombinant plasmid pAd-SOCS3 was obtained by homologous recombination between pAdTrack-CMV-SOCS3 and the adenoviral backbone plasmid pAdEasy-1 in BJ5183. The pAd-SOCS3 was linearized by Pac I and transfected into HEK293 cells via liposome. The recombinant adenovirus was packaged and amplified in HEK293 cells. After purifying, virus titer was determined by tissue culture infectious dose 50 (TCID50). Using the recombinant adenoviruses to infect porcine primary adipocytes, the expression of green fluorescent protein (GFP) was observed by fluorescent microscopy, and SOCS3 gene was identified by RT-PCR and Western blotting. Restriction enzyme and PCR analysis demonstrated that the recombinant adenovirus vector was constructed correctly, and the virus titer reached 1.2x10(9) PFU/mL. The result of RT-PCR and Western blotting showed that SOCS3 mRNA and protein expression was remarkably increased in porcine primary adipocytes infected with recombinant adenovirus. In conclusion, this study successfully constructed the recombinant adenovirus containing SOCS3 gene, and can be helpful for further research on the function of SOCS3.
