Nitric oxide inducing function and intracellular movement of chicken interleukin-18 in cultured cells.
To evaluate the characteristics of chicken interleukin-18 (ChIL-18) in different forms in vitro, the ChIL-18 full-length gene (ChIL-18-F) and the ChIL-18 presumed mature protein gene (ChIL-18-M) were cloned and inserted into the eukaryotic expression vector pCI, to construct recombinant pCI-ChIL-18-F and pCI-ChIL-18-M. The recombinant plasmids were then transferred into chicken splenic lymphocytes (CSLs). Western blot showed that ChIL-18-F, with a molecular weight of 23.0 kDa, was produced in CSLs transfected by pCI-ChIL-18-F; ChIL-18-M, with a molecular weight of 19.5 kDa, was produced in CSLs transfected by pCI-ChIL-18-M. The nitric oxide (NO) level in the transfected CSLs and the culture medium at different time points was further examined under confocal microscopy using 4,5-diaminofluorescein staining. The results showed that both pCI-ChIL-18-F and pCI-ChIL-18-M groups showed significant increase in intracellular and extracellular NO production compared with pCI transfected control cells. These results suggest that both ChIL-18-F and ChIL-18-M could stimulate NO secretion in CSLs. To characterize the intracellular distribution of ChIL-18, ChIL-18-F and ChIL-18-M were each fused to the enhanced green fluorescent protein gene, and expressed in Vero cells. The results showed that the ChIL-18-F tended to the membranous region in Vero cells, while ChIL-18-M did not. This indicates that the N-terminal 27 amino acid peptide helped ChIL-18 target to Vero cell membranes.
Xu
Jian
J
Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310029, China.
Deng
Tong-Le
TL
Li
Long
L
You
Zhen-Qiang
ZQ
Wan
Wang-Jun
WJ
Yu
Lian
L
D000818:Animals; D002478:Cells, Cultured; D002645:Chickens; D002522:Chlorocebus aethiops; D020382:Interleukin-18 / Q000502:physiology*; D008214:Lymphocytes / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000494:pharmacology; D013154:Spleen / Q000166:cytology; D014162:Transfection; D014709:Vero Cells
D020382:Interleukin-18; D011994:Recombinant Proteins; D009569:Nitric Oxide