Molecular cloning, characterization and biological activity of duck interleukin-17F

다국어 초록 (Multilingual Abstract)

Interleukin-17F (IL-17F) is a proinflammatory cytokine, which plays an important role in gut homeostasis. A full-length duck IL-17F (duIL-17F) cDNA with a 501-bp coding region was isolated from ConA-stimulated duck splenic lymphocytes. The duIL-17F is predicted to encode 166 amino acids, which include a 26-amino acid signal peptide, single N-linked glycosylation site, and six cysteine residues that are conserved in mammalian IL-17 family. The duIL-17F shares 77.5% amino acid sequence identity with chicken IL-17F (chIL-17F), 37-46% with corresponding mammalian homologues and 53.5% with previously described duck IL-17A (duIL-17A). The duIL-17F transcript was preferentially expressed in a wide range of normal tissues; highest level was detected in the liver and moderate levels were found in thymus, bursa, kidney, and intestinal tissues. Expression levels of duIL-17F transcript were slightly up-regulated in ConA- and LPS-activated splenic lymphocytes, but not in poly I:C stimulated cells. The recombinant duIL-17F, like duIL-17A, induced IL-1β, IL-6, and IL-8 in primary duck embryonic fibroblasts (DEFs), suggesting duIL-17F involvement in inflammatory response and thus, can function as a proinflammatory cytokine. Moreover, duIL-17F directly interacts with duIL-17A and forms a heterodimer. In Salmonella-infected tissues, only expression levels of duIL-17A, but not duIL-17F, were significantly up-regulated in the liver and spleen compared with those of uninfected controls. Thus, further analysis of the contributions of IL-17F and IL-17A to other Salmonella spp. and disease models remains to be elucidated to expand our understanding of its biological functions.

목차 (Table of Contents)

• 1. Introduction 1
• 2. Materials and Methods 4
• 2.1. Animals and infection 4
• 2.2. Molecular cloning of duIL-17F cDNA 4
• 2.3. Sequence analysis 5
• 2.4. Cell culture 5
• 2.5. Plasmid construction and cell transfection 6
• 2.6. Western blot analysis 7
• 2.7. Immunoprecipitation 7
• 2.8. Biological activity of recombinant duIL-17F 8
• 2.9. Quantitative real-time RT-PCR 8
• 2.10. Statistical analysis 11
• 3. Results 12
• 3.1. Cloning and characterization of duIL-17F cDNA 12
• 3.2. Distribution of duIL-17F expression in uninfected tissues and in mitogen-stimulated lymphocytes 18
• 3.3. Molecular weight and biological activity of recombinant duIL-17F 21
• 3.4. Interaction between duIL-17A and duIL-17F 24
• 3.5. Quantitative analysis of duIL-17A and duIL-17F mRNA expression levels in Salmonella-infected ducks 26
• 4. Discussion 29
• References 33