Cloning, in vitro expression and bioactivity of duck interleukin-18.
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.
Chen
Hong-Ying
HY
College of Animal Husbandry and Veterinary, Henan Agricultural University, Zhengzhou 450002, China.
Cui
Bao-An
BA
Xia
Ping-An
PA
Li
Xin-Sheng
XS
Hu
Gong-Zheng
GZ
Yang
Ming-Fan
MF
Zhang
Hong-Ying
HY
Wang
Xue-Bin
XB
Cao
Su-Fang
SF
Zhang
Long-Xian
LX
Kang
Xiang-Tao
XT
Tu
Ke
K
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology / Q000494:pharmacology; D001483:Base Sequence; D015153:Blotting, Western / Q000662:veterinary; D002645:Chickens; D003001:Cloning, Molecular; D004372:Ducks / Q000235:genetics* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D004926:Escherichia coli / Q000235:genetics; D053124:Influenza A Virus, H5N1 Subtype / Q000276:immunology; D007252:Influenza Vaccines / Q000276:immunology / Q000494:pharmacology; D020382:Interleukin-18 / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000187:drug effects / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010802:Phylogeny; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D016415:Sequence Alignment
D000911:Antibodies, Monoclonal; D007252:Influenza Vaccines; D020382:Interleukin-18; D011994:Recombinant Proteins