Interleukin-12 (IL-12) is a pleiotropic
cytokine which bridges innate and adaptive immunity in defense against pathogens.
IL-12 proved to be an effective and successful adjuvant to enhance both the innate and adaptive immune responses and could be applicable for a rationale
vaccine formulation in fish against pathogen
infection. We have cloned the p35 and p40 cDNAs of
IL-12 from orange-spotted grouper (Epinephelus coioides). Grouper
IL-12 most resembles with sea bass orthologues; moderate to low identity with other teleost and mammalian counterparts. The structural model of grouper
IL-12 heterodimer revealed NC(141)F three
amino acid patch of grouper p35, which is present in teleost p35 but absent in mammalian and avian p35, and is spatially nearby the conserved
cysteine residue located at A-helix of p35 to form a
disulfide bond when the 14aa
peptide located at loop 1 of grouper p35 was aligned with human corresponding exon 4, instead of exon 5. The results indicated that the loss of this 3aa patch during evolution was compensated by the duplication of exon 4 in mammalian p35 to gain another
cysteine residue to form a
disulfide bond, evidenced by chicken p35 which does not contain NCF corresponding 3-aa patch nor exon 4 duplication. Accordingly, the inter-chain
disulfide bond of
IL-12 heterodimer is conserved from teleost to mammalian
IL-12. A single chain grouper
IL-12 (scgIL-12) construct linked by (G4S)3 was successfully expressed in baculovirus-insect cell system; its identity has been confirmed by LC/MS/MS. In addition, the
biological activity of recombinant scgIL-12 (rscgIL-12) are demonstrated for its stimulation of PBL proliferation, chemotactic migration, induction of TNF-α gene expression and a plausible adjuvant effect of prolonged protection against
parasite infection in fish. We illustrated the first time in lower vertebrate that grouper
IL-12 possesses both
cytokine and
chemokine activities.