Functional Identification Of Grass Carp Interleukin-6 And Its Receptors

Interleukin-6(IL-6)is a pleiotropic cytokine that plays a crucial role in immune system,nervous system,liver regeneration,and the metabolic control of body.IL-6 firstly binds to its specific IL-6 receptor(IL-6R)and then associates with the second receptor protein,gp130,forming a trimer to initiate intracellular signaling.The functions of IL-6 and its receptors system have been extensively studied in mammalian immune system but not in bony fish.Using grass carp as the model,we obtained the recombinant grass carp IL-6 protein as a tool to study the properties of IL-6R and its roles in inflammation.Because IL-6R has two forms as membrane binding form and soluble form(sIL-6R),we also explored the properties of sIL-6R through recombinant expression of it.We obtained the recombinant protein of grass carp IL-6(rgcIL-6)by the Easy Select? Pichia Expression Kit and the bioactivity of rgcIL-6 was verified.We cloned the full length cDNA of grass carp IL-6R with 2223 bp containing an ORF of 1785 bp encoding 594 amino acids.Multiple alignment of the amino acid sequence of grass carp IL-6R with those of other species showed that the identities were low(38.03%-45%).Tissue distribution of IL-6R showed that the highest expression of IL-6R was found in head kidney.Besides,we obtained the recombinant protein of grass carp sIL-6R(rgcs IL-6R)by a Escherichia coli expression system followed by inclusion body renaturation.Moreover,intraperitoneal injection of A.hydrophila up-regulated the expression of IL-6R protein in head and trunk kidney.In addition,LPS and poly I:C up-regulated the expression of IL-6R mRNA in head kidney leukocytes(HKL),and the latter promoted gcs IL-6R protein generation.Finally,the binding of rgc IL-6 and rgcsIL-6R was demonstrated,and the in vitro experiment showed rgcsIL-6R could enhance the effects of IL-6 on TNF-?,IL-10 and SOCS3 mRNA expression in HKL.Taken these together,IL-6R plays a crucial role in bacteria infection in grass carp.Using the rgcIL-6 and rgcsIL-6R protein,we found rgcs IL-6R could enhance the immune function of IL-6 in vitro.This is the first time to explore the regulation of IL-6R in inflammation and reveal the effect of s IL-6R on IL-6's immune function in bony fish.This provides the clues for preventing grass carp from diseases.