Key Laboratory of Molecular Microbiology and Biotechnology (Ministry of Education), College of Life Sciences, Nankai University, Tianjin, 300071, People's Republic of China.
Vet Res Commun. 2009 Oct;33(7):723-33. doi: 10.1007/s11259-009-9221-8. Epub 2009 Apr 15.
Fetal bovine lung (FBL) cells are used in the culture of viruses which infect cattle and ISG15 plays a role in innate immunity against viral infections. However, whether the expression of ISG15 gene can be induced in FBL cells is still unknown. In this work, the expression of ISG15 in cultured FBL cells was detected after stimulated with poly I:C or LPS. Real-time PCR analyses revealed that the transcript of ISG15 can be induced by poly I:C or LPS. The increased expression of free ISG15 was confirmed via Western blotting. Furthermore, immunofluorescence assays demonstrated that IRF-3 was translocated from the cytoplasm to the nucleus in the FBL cells treated with poly I:C. Chromatin immunoprecipitation assays showed that IRF-3 can bind to the promoter of the bISG15 gene. To demonstrate IRF-3 can promote the expression of bISG15, we establish a luciferase-reporter system of bovine ISG15 gene in 293 T cells. The luciferase assay showed that the over-expression of bovine IRF-3 could activate the promoter of bISG15 gene. Taken together, these results suggest that the expression of bISG15 can be induced in FBL cells stimulated with poly I:C or LPS, and IRF-3 may play a role in inducing the expression of ISG15 in FBL cells.
胎牛肺（FBL）细胞用于培养感染牛的病毒，ISG15 在抗病毒感染的先天免疫中发挥作用。然而，FBL 细胞中 ISG15 基因的表达是否可以被诱导尚不清楚。在这项工作中，用 poly I:C 或 LPS 刺激培养的 FBL 细胞后，检测了 ISG15 的表达。实时 PCR 分析显示，poly I:C 或 LPS 可诱导 ISG15 的转录。通过 Western blot 证实了游离 ISG15 的表达增加。此外，免疫荧光分析表明，poly I:C 处理的 FBL 细胞中 IRF-3 从细胞质易位到细胞核。染色质免疫沉淀分析表明，IRF-3 可结合 bISG15 基因的启动子。为了证明 IRF-3 可以促进 bISG15 的表达，我们在 293T 细胞中建立了牛 ISG15 基因的荧光素酶报告基因系统。荧光素酶测定表明，牛 IRF-3 的过表达可以激活 bISG15 基因的启动子。总之，这些结果表明，poly I:C 或 LPS 刺激的 FBL 细胞中可以诱导 bISG15 的表达，IRF-3 可能在诱导 FBL 细胞中 ISG15 的表达中发挥作用。
