Cloning, in vitro expression and bioactivity of duck interleukin-18
- 作者：Hong-Ying Chen ; Bao-An Cui ; Ping-An Xia ; Xin-Sheng Li ; Gong-Zheng Hu ; Ming-Fan Yang ; Hong-Ying Zhang ; Xue-Bin Wang ; Su-fang Cao ; Long-Xian Zhang ; Xiang-Tao Kang ; Ke Tu
- 关键词：Duck ; Interleukin-18 ; In vitro expression ; In vivo bioactivity
- 刊名：Veterinary Immunology and Immunopathology
- 出版年：2008
- 期刊代码：105_01652427
- 类别：abs
- 出版时间：15 June 2008
- 卷：123
- 期：3-4
- 页码：205-214
- 文件大小：767 K
摘要
The encoding sequence for duck IL-18 was obtained, using reverse transcription-polymerase chain reaction, from mRNA harvested from Con A-stimulated Gushi (GS) duck splenic mononuclear cells. Recombinant duck IL-18 (rduIL-18) was produced in a prokaryotic expression system. In vitro bioactivity of rduIL-18 was determined in a lymphocyte proliferation assay and in vivo bioactivity of rduIL-18 was assessed by addition to a vaccine. Monoclonal antibody (mAb) and polyclonal antibodies (pAbs) specific for rduIL-18 were generated and subsequently characterized by ELISA, Western blot and neutralizing assays. Sequence analysis of GS duck IL-18 demonstrated an open reading frame (ORF) of 603 base pairs encoding for a 200 amino acid precursor protein. The duck encoding sequence shares 85.3%similarity to the chicken equivalent, at the nucleotide level. A His-duIL-18 fusion protein was recognized in Western blot by mAbs against duck and chicken IL-18 (chIL-18), but not by mAb against human IL-18. Recombinant duIL-18 induced in vitro proliferation of Con A-stimulated duck splenocytes and enhanced the immune response of ducks vaccinated with an inactivated oil emulsion vaccine against avian influenza virus. PAb and mAb 5B2 against rduIL-18 had neutralizing ability, inhibiting the biological activities of both recombinant duIL-18 and endogenous duIL-18. The results indicate that rduIL-18 has the potential to be used as an immunoadjuvant, and the mAb against rduIL-18 further facilitates basic immunobiological studies of the role of IL-18 in the avian immune system.