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Construction of an expression system for bioactive IL-18 and generation of recombinant canine distemper virus expressing IL-18.

Liu Y, Sato H, Hamana M, Moonan NA, Yoneda M, Xia X, Kai C - J. Vet. Med. Sci. (2014)

Bottom Line: However, IL-18 protein lacks a typical signal sequence and requires cleavage into its mature active form by caspase 1.The expressed proIL-18 proteins were processed to their mature forms in the cells.These results indicate that the signal sequence from hIL-2 is suitable for the secretion of mature IL-18.

View Article: PubMed Central - PubMed

Affiliation: Laboratory Animal Research Center, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

ABSTRACT
Interleukin 18 (IL-18) plays an important role in the T-helper-cell type 1 immune response against intracellular parasites, bacteria and viral infections. It has been widely used as an adjuvant for vaccines and as an anticancer agent. However, IL-18 protein lacks a typical signal sequence and requires cleavage into its mature active form by caspase 1. In this study, we constructed mammalian expression vectors carrying cDNA encoding mature canine IL-18 (cIL-18) or mouse IL-18 (mIL-18) fused to the human IL-2 (hIL-2) signal sequence. The expressed proIL-18 proteins were processed to their mature forms in the cells. The supernatants of cells transfected with these plasmids induced high interferon-γ production in canine peripheral blood mononuclear cells or mouse splenocytes, respectively, indicating the secretion of bioactive IL-18. Using reverse genetics, we also generated a recombinant canine distemper virus that expresses cIL-18 or mIL-18 fused to the hIL-2 signal sequence. As expected, both recombinant viruses produced mature IL-18 in the infected cells, which secreted bioactive IL-18. These results indicate that the signal sequence from hIL-2 is suitable for the secretion of mature IL-18. These recombinant viruses can also potentially be used as immunoadjuvants and agents for anticancer therapies in vivo.

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Mammalian expression plasmids for canine and mouse IL-18. (A) Schematicrepresentation of IL-18 constructs. From top to bottom: full-length sequence of canineIL-18, mature canine IL-18 fused to the canine IL-12 signal sequence, mature canineIL-18 fused to the human IL-2 signal sequence and mature mouse IL-18 fused to thehuman IL-2 signal sequence were inserted into the pCAGGS and pFuse–hIgG2–Fc2 vectors.(B) The expression of IL-18 by transfected 293T cells was analyzed with immunoblottingusing anti-GAPDH, anti-cIL-18 and anti-mIL-18 antibodies. 1. Mock; 2. pCAG–cIL18; 3.pCAG–cIL12ss–cIL18; 4. pFuse–hIL2ss–cIL18; 5. pFuse–hIL2ss–mIL18.
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fig_001: Mammalian expression plasmids for canine and mouse IL-18. (A) Schematicrepresentation of IL-18 constructs. From top to bottom: full-length sequence of canineIL-18, mature canine IL-18 fused to the canine IL-12 signal sequence, mature canineIL-18 fused to the human IL-2 signal sequence and mature mouse IL-18 fused to thehuman IL-2 signal sequence were inserted into the pCAGGS and pFuse–hIgG2–Fc2 vectors.(B) The expression of IL-18 by transfected 293T cells was analyzed with immunoblottingusing anti-GAPDH, anti-cIL-18 and anti-mIL-18 antibodies. 1. Mock; 2. pCAG–cIL18; 3.pCAG–cIL12ss–cIL18; 4. pFuse–hIL2ss–cIL18; 5. pFuse–hIL2ss–mIL18.

Mentions: Bioactivity of recombinant IL-18 with different signal sequences in mammaliancells: First, we compared the secretion efficiency of the signal sequence withthat of mature cIL-18. 293T cells were transfected with recombinant plasmids encoding entirecIL-18 (pCAG–cIL18) or mature cIL-18 fused to the cIL-12 signal sequence(pCAG–cIL12ss–cIL18) or to the human IL-2 signal sequence (pFuse–hIL2ss–cIL18) (Fig. 1AFig. 1.


Construction of an expression system for bioactive IL-18 and generation of recombinant canine distemper virus expressing IL-18.

Liu Y, Sato H, Hamana M, Moonan NA, Yoneda M, Xia X, Kai C - J. Vet. Med. Sci. (2014)

Mammalian expression plasmids for canine and mouse IL-18. (A) Schematicrepresentation of IL-18 constructs. From top to bottom: full-length sequence of canineIL-18, mature canine IL-18 fused to the canine IL-12 signal sequence, mature canineIL-18 fused to the human IL-2 signal sequence and mature mouse IL-18 fused to thehuman IL-2 signal sequence were inserted into the pCAGGS and pFuse–hIgG2–Fc2 vectors.(B) The expression of IL-18 by transfected 293T cells was analyzed with immunoblottingusing anti-GAPDH, anti-cIL-18 and anti-mIL-18 antibodies. 1. Mock; 2. pCAG–cIL18; 3.pCAG–cIL12ss–cIL18; 4. pFuse–hIL2ss–cIL18; 5. pFuse–hIL2ss–mIL18.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4197152&req=5

fig_001: Mammalian expression plasmids for canine and mouse IL-18. (A) Schematicrepresentation of IL-18 constructs. From top to bottom: full-length sequence of canineIL-18, mature canine IL-18 fused to the canine IL-12 signal sequence, mature canineIL-18 fused to the human IL-2 signal sequence and mature mouse IL-18 fused to thehuman IL-2 signal sequence were inserted into the pCAGGS and pFuse–hIgG2–Fc2 vectors.(B) The expression of IL-18 by transfected 293T cells was analyzed with immunoblottingusing anti-GAPDH, anti-cIL-18 and anti-mIL-18 antibodies. 1. Mock; 2. pCAG–cIL18; 3.pCAG–cIL12ss–cIL18; 4. pFuse–hIL2ss–cIL18; 5. pFuse–hIL2ss–mIL18.
Mentions: Bioactivity of recombinant IL-18 with different signal sequences in mammaliancells: First, we compared the secretion efficiency of the signal sequence withthat of mature cIL-18. 293T cells were transfected with recombinant plasmids encoding entirecIL-18 (pCAG–cIL18) or mature cIL-18 fused to the cIL-12 signal sequence(pCAG–cIL12ss–cIL18) or to the human IL-2 signal sequence (pFuse–hIL2ss–cIL18) (Fig. 1AFig. 1.

Bottom Line: However, IL-18 protein lacks a typical signal sequence and requires cleavage into its mature active form by caspase 1.The expressed proIL-18 proteins were processed to their mature forms in the cells.These results indicate that the signal sequence from hIL-2 is suitable for the secretion of mature IL-18.

View Article: PubMed Central - PubMed

Affiliation: Laboratory Animal Research Center, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

ABSTRACT
Interleukin 18 (IL-18) plays an important role in the T-helper-cell type 1 immune response against intracellular parasites, bacteria and viral infections. It has been widely used as an adjuvant for vaccines and as an anticancer agent. However, IL-18 protein lacks a typical signal sequence and requires cleavage into its mature active form by caspase 1. In this study, we constructed mammalian expression vectors carrying cDNA encoding mature canine IL-18 (cIL-18) or mouse IL-18 (mIL-18) fused to the human IL-2 (hIL-2) signal sequence. The expressed proIL-18 proteins were processed to their mature forms in the cells. The supernatants of cells transfected with these plasmids induced high interferon-γ production in canine peripheral blood mononuclear cells or mouse splenocytes, respectively, indicating the secretion of bioactive IL-18. Using reverse genetics, we also generated a recombinant canine distemper virus that expresses cIL-18 or mIL-18 fused to the hIL-2 signal sequence. As expected, both recombinant viruses produced mature IL-18 in the infected cells, which secreted bioactive IL-18. These results indicate that the signal sequence from hIL-2 is suitable for the secretion of mature IL-18. These recombinant viruses can also potentially be used as immunoadjuvants and agents for anticancer therapies in vivo.

Show MeSH
Related in: MedlinePlus