Hu Zhonglan, Chen Zhenyu, Huang Nongyu, Teng Xiu, Zhang Jun, Wang Zhen, Wei Xiaoqiong, Qin Ke, Liu Xiao, Wu Xueping, Tang Huan, Zhu Xiaofeng, Cui Kaijun, Li Jiong
State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China.
State Key Laboratories of Agrobiotechnology, College of Biological Science, China Agricultural University, Beijing 100193, People's Republic of China.
Protein Expr Purif. 2015 Mar;107:76-82. doi: 10.1016/j.pep.2014.10.016. Epub 2014 Nov 6.
Members of the interleukin-1 (IL-1) family play important roles in inflammation and host defense against pathogens. Here, we describe a novel member of the IL-1 family, interleukin-38 (IL-38, IL-1F10, or IL-1HY2), which was discovered in 2001. Although the functional role of IL-38 remains unclear, recent reports show that IL-38 binds to the IL-36 receptor (IL-36R) which is also targeted by the IL-36 receptor antagonist (IL-36Ra). Consequently, these two molecules have similar effects on immune cells. Here, we describe the expression of soluble and active recombinant IL-38 in Escherichia coli (E. coli). The IL-38 gene sequence was optimized for expression in E. coli and then cloned into a pEHISTEV expression vector, which has an N-terminal 6-His affinity tag under control of the T7 lac strong promoter. Optimization of culture conditions allowed induction of the recombinant fusion protein with 0.1 mM isopropyl β-D-1-thio galactoside (IPTG) at 37°C for 4h. The recombinant fusion protein was purified using an Ni affinity column and was further digested with TEV protease; the cleaved protein was purified by molecular-exclusion chromatography. Next, we measured IL-38 binding ability using functional ELISA. The purified proteins were used to immunize a New Zealand white rabbit four times to enable the production of polyclonal antibodies. The specificity of the prepared polyclonal antibodies was determined using Western blot, and the results showed they have high specificity against IL-38. Here, we describe the development of an effective and reliable method to express and purify IL-38 and anti-IL-38 antibodies. This will enable the function and structure of IL-38 to be determined.
白细胞介素-1（IL-1）家族成员在炎症反应和宿主抵御病原体过程中发挥重要作用。在此，我们描述了IL-1家族的一个新成员——白细胞介素-38（IL-38，IL-1F10或IL-1HY2），它于2001年被发现。尽管IL-38的功能作用尚不清楚，但最近的报道显示，IL-38与白细胞介素-36受体（IL-36R）结合，而IL-36受体拮抗剂（IL-36Ra）也作用于该受体。因此，这两种分子对免疫细胞具有相似的作用。在此，我们描述了可溶性活性重组IL-38在大肠杆菌中的表达。对IL-38基因序列进行优化以使其在大肠杆菌中表达，然后将其克隆到pEHISTEV表达载体中，该载体在T7 lac强启动子控制下带有N端6-组氨酸亲和标签。通过优化培养条件，可在37°C用0.1 mM异丙基β-D-1-硫代半乳糖苷（IPTG）诱导重组融合蛋白表达4小时。重组融合蛋白用镍亲和柱纯化，并用TEV蛋白酶进一步消化；切割后的蛋白通过分子排阻色谱法纯化。接下来，我们使用功能性酶联免疫吸附测定（ELISA）检测IL-38的结合能力。用纯化的蛋白对新西兰白兔进行四次免疫以产生多克隆抗体。使用蛋白质免疫印迹法确定所制备多克隆抗体的特异性，结果表明它们对IL-38具有高特异性。在此，我们描述了一种有效且可靠的表达和纯化IL-38及抗IL-38抗体的方法。这将有助于确定IL-38的功能和结构。
