Yuan X L, Li Y, Pan X H, Zhou M, Gao Q Y, Li M C
Zhejiang Provincial Key Laboratory of Pathophysiology, Department of Immunology, Ningbo University School of Medicine, Ningbo, 315211, China.
Mol Biol (Mosk). 2016 May-Jun;50(3):466-73. doi: 10.7868/S0026898416030137.
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.
白细胞介素（IL）-38是IL-1细胞因子家族的最新成员。然而，由于缺乏有效方法来大量产生IL-38，其确切功能了解甚少。在本研究中，描述了重组人IL-38的克隆、表达、纯化及活性分析。将人IL-38 cDNA克隆到原核表达载体pET-44中。含C-六聚组氨酸标签的重组IL-38在异丙基-β-D-硫代半乳糖苷诱导的大肠杆菌BL21（DE3）中表达。表达的融合蛋白通过Ni-NTA亲和层析纯化。IL-38蛋白主要存在于可溶部分。纯化的IL-38在SDS-PAGE上呈现单一条带，从1 L细菌培养物中获得的IL-38产量为4 mg，纯度超过98%，内毒素水平低（<0.1 EU/μg）。蛋白质免疫印迹法证实了纯化蛋白的身份。活性分析表明，IL-38能有效抑制脂多糖激活的THP-1细胞中促炎细胞因子如肿瘤坏死因子-α、IL-1β、IL-17和单核细胞趋化蛋白-1的表达。具有生物活性的IL-38的产生和特性研究将有助于其在临床应用中的潜在作用。
