Preparation of vector of zinc finger protein A20 gene silence and its preliminary application
- VernacularTitle:锌指蛋白A20基因沉默载体制备及初步应用
- Author:
Lijuan WU
;
Ping CHEN
;
Gefei KANG
;
Jianxin JIANG
;
Peifang ZHU
- Publication Type:Journal Article
- Keywords:
Zinc finger;
Lipopolysaccharide;
Gene silence;
Real-time polymerasechain reaction
- From:
Chinese Journal of Trauma
2008;24(4):294-297
- CountryChina
- Language:Chinese
-
Abstract:
Objective To design and prepare an RNA interfering vector for effectively inhibiting the cellular expression of zinc finger protein A20 and observe the effect of A20 gene silence on cellular inflammatory response. Methods Specific RNA interfering oligonucleotide fragments (ASRF) were designed and synthesized artificially and the A20 RNA interfering vector pSUPER-EGFP-A20 siRNA constructed. Human monocyte cell line THP1 was used to infect the pSUPER-EGFP-A20 siRNA by means of genetic transfection technique; then, silence rate of cellular A20 was analyzed by real-time polymerase chain reaction (PCR). In the meantime, the activity of nuclear transcription factor nuclear factor-κB (NF-κB) and the level of tumor necrosis factor-α (TNF-α) in culture supernatant were measured by ELISA. Results Of two specific inhibitory oligonueleotide fragments of A20, the fragment M59465-385R/F had a higher inhibition to A20 expression, with rate of A20 gene silence of 83.86%. Preliminary application showed that after A20 gene silence, the activity of NF-κB was increased by 78.13% and the level of TNF-α in cell culture supernatant was increased by 49.30%. Conclusions Vector of A20gene silence with a high efficiency is obtained successfully. Preliminary application indicates that the expression of A20 can down-regulate the degree of cellular inflammatory responses.
