Interleukin (IL)-38 is the latest member of the IL-1 cytokine family. However, as a result of lacking efficient method to generate relatively large quantity of IL-38, its precise functions are poorly understood. In the present study, the cloning, expression, purification, and activity analysis of recombinant human IL-38 was described. Human IL-38 cDNA was cloned into the prokaryotic expression vector pET-44. The recombinant IL-38 containing a C-hexahistidine tag was expressed in Escherichia coli BL21 (DE3) which induced by isopropyl-β-D-thiogalactoside. The expressed fusion protein was purified by Ni-NTA affinity chromatography. IL-38 protein was largely found in the soluble fraction. The purified IL-38 appeared a single band on SDS-PAGE, the yield of IL-38 was 4 mg from 1 L of bacterial culture, and the purity was more than 98% with low endotoxin level (<0.1 EU/μg). Western blotting confirmed the identity of the purified protein. Activity analysis showed that IL-38 can inhibit effectively the expression of proinflammatory cytokines, such as tumor necrosis factor-α, IL-1β, IL-17, and monocyte chemoattractant protein-1 in lipopolysaccharide-activated THP-1 cells. The production and characterization of biologically active IL-38 will be beneficial for its potential role in clinical applications.

An 8-week feeding trial was conducted to determine the dietary leucine requirement for juvenile swimming crabs reared in cement pools. Six isonitrogenous and isolipidic practical diets (430 g/kg crude protein and 70 g/kg crude lipid) were formulated to contain graded leucine levels which ranged from 16.7 to 26.7 g/kg (dry weight). Each diet was randomly assigned to triplicate groups of 60 juvenile swimming crabs (initial average weight 3.75 ± 0.12 g) that were stocked in rectangle plastic baskets. The results of the present study indicated that dietary leucine levels significantly influenced weight gain (WG) and specific growth ratio (SGR) (p < .05), crab fed the diet containing 22.7 g/kg leucine had significantly higher WG and SGR than those fed the other diets.Feed efficiency and protein efficiency ratio were not significantly affected by the dietary leucine levels (p > .05). Total protein, cholesterol, triglyceride and glucose in serum were significantly affected by the dietary leucine levels. Aspartate aminotransferase (AST) and alanine aminotransferase activities in hemolymph, AST and superoxide dismutase activities in hepatopancreas were significantly affected by dietary leucine levels; moreover, crab fed the 16.7 g/kg leucine diet had higher malondialdehyde in hemolymph and hepatopancreas than those fed the other diets. Crab fed the diet containing 24.9 g/kg leucine had higher phenoloxidase activity in hemolymph than those fed the other diets. Based on two-slope broken-line model of SGR against dietary leucine levels, the optimal dietary leucine requirement for growth was estimated to be 22.1 g/kg of the dry diet (corresponding to 51.4 g/kg of dietary protein on a dry weight basis). In summary, findings of this study indicated that dietary leucine could improve growth performance and antioxidant status. K E Y W O R D S enzyme activities, growth performance, leucine level, Portunus trituberculatus

Interferon (IFN)-λ3, a member of the type III IFN family, is a pleiotropic cytokine that exhibits potent antiproliferative, antiviral, and immunoregulatory activities. For further functional study of IFN-λ3, we developed an efficient procedure that includes cloning, expression, and purification to obtain relatively large quantity of mouse IFN-λ3 fusion protein. The mature IFN-λ3 protein-coding region was cloned into the prokaryotic expression vector pET-44. IFN-λ3 contains a hexahistidine tag at its C-terminus. We used Ni(2+)-nitrilotriacetic acid agarose-affinity chromatography to purify the expressed soluble protein. The purified IFN-λ3 inhibited significantly IL-13 production in stimulated RAW264.7 macrophages. Our findings show that the production of soluble IFN-λ3 proteins by the pET-44 vector in Escherichia coli is a good alternative for the production of native IFN-λ3 and could be useful for the production of other IFN proteins.

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