摘要
为了研究布鲁菌感染机体后锌指蛋白A20调节天然免疫的机制,我们尝试构建A20表达的巨噬细胞模型。通过分子生物学手段构建表达A20的慢病毒;Western blot检测A20的表达;以内毒素(LPS)刺激细胞,Western blot检测IκB-α降解变化且ELISA检测炎性细胞因子TNF-α及IL-6的水平来判定A20表达细胞模型是否成功。限制性内切酶酶切鉴定、DNA测序及慢病毒包装试验表明A20重组慢病毒构建成功;Western blot证实感染J774A.1后A20蛋白表达明显增加;A20表达的J774A.1受到LPS刺激后IκB-α未发生降解,TNF-α及IL-6水平明显低于空载体及空白对照组(P<0.01)。结果表明,本研究成功构建了A20表达的巨噬细胞模型。
The macrophage J774A. 1 cell line model of A20 expression was constructed to future investigates the role of A20 in the regulation of innate immunity after cells were infected with Brucella. To meet the aim,J774A. 1 cells were infected with lentivirus containing A20. The ex- pression of protein A20 was detected by Western blot. The expression of IκB-α was detected in J774A. 1 cells after LPS treatment by Western blot and the concentrations of TNFα,IL- 6 in the supernatants were determined by cytokine ELISA kits. Our data indicated that A20 was expressed in J774A. 1 cells. The degradation IκB-α was abrogated in response to LPS stimulation in the A20 over-expression J774A. 1 cells. The productions of inflammatory cytokine TNF-α, IL-6 were de- creased significantly compared with non-expression A20 J774A. 1 cells. Thus, the macrophage J774A. 1 cell line model of A20 expression was successfully constructed.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2014年第3期411-414,421,共5页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目(31372409)
