Isolation And Identification Of Interleukin 22,Its Receptor And Binding Protein From Grass Carp

Interleukin 22(IL-22)is a member of the IL-10 family.As a multifunctional cytokine,it plays an important role in inflammatory diseases.In generally,a proper amount of IL-22 can protect the body by inhibiting inflammation,possessing antibacterial activity,repairing mucosal damage,and promoting cell regeneration.However,excessive IL-22 can cause functional disorders and autoimmune diseases.IL-22RA1 is the IL-22 receptor,while IL-22 BP is the binding protein of IL-22.Increasing attentions have been paid the relationship and functional roles of IL-22,IL-22RA1 and IL-22 BP in mammals,but their information is still limited in teleost.In this study,using grass carp as a research model,IL-22 was isolated and cloned.Its CDS sequence contained 510 nucleotides and encoded 169 amino acids.The multiple alignment assay of amino acid sequence revealed that it shared 14.77%-39.91% similarity compared with homologs from other species.Moreover,a prokaryotic expression system,renaturation treament of inclusion body protein,and eukaryotic expression system were used to obtain recombinant IL-22 protein and verify its biological activity.In addition,using the validated IL-22 polyantibody,the stimulation of PMA on IL-22 secretion was detected in grass carp headkidney leukocytes.Subsequently,gene localization analysis isolated and identified grass carp IL-22RA1,which contained 1533 nucleotides and encoded 510 amino acids.Finally,IL-22 BP was isolated and cloned,and its CDS sequence was 702 bp in length and encoded a total of 233 amino acids.Grass carp IL-22 BP amino acid sequence shared 31.20% to 82.83% similarity with that from other species.Tissue distribution assay of grass carp IL-22 BP showed its major expression in small intestine and liver,and less distribution in head kidney,kidney,spleen,and gill.In grass carp infected with Aeromonas hydrophila,the expression of IL-22 BP was significantly reduced in the tested tissue,particulary in intestine and liver.These findings laid the foundation for further exploring the role of the relationship among IL-22,IL-22RA1,and IL-22 BP in grass carp immunity.