Pöhlmann Christoph, Brandt Manuela, Mottok Dorothea S, Zschüttig Anke, Campbell John W, Blattner Frederick R, Frisch David, Gunzer Florian
Institute of Medical Microbiology and Hygiene, Dresden University of Technology, Dresden, Germany.
J Mol Microbiol Biotechnol. 2012;22(1):1-9. doi: 10.1159/000336043. Epub 2012 Feb 21.
Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine, with therapeutic applications in inflammatory bowel disease. For the in situ delivery of IL-10 by Escherichia coli as carrier chassis, a modified transporter was designed with the ability to secrete biologically active IL-10. De novo DNA synthesis comprised a 561-bp fragment encoding the signal sequence of the E. coli outer membrane protein F fused in frame to an E. coli codon-optimized mature human IL-10 gene under control of a T7 promoter. The construct was overexpressed in E. coli laboratory strains, E. coli BL21 (DE3) and E. coli MDS42:T7. The mean concentrations of human IL-10 in the periplasm and culture supernatant of E. coli BL21 (DE3) were 355.8 ± 86.3 and 5.7 ± 1.7 ng/ml, respectively. The molecular mass of the recombinant E. coli-derived human IL-10 was 19 kDa, while under non-reducing conditions the native IL-10 dimer could be demonstrated. Reduction of tumor necrosis factor-α secretion in lipopolysaccharide-stimulated mouse macrophages and detection of the activated form of the transcription factor signal transducer and activator of transcription protein 3 proved the biological activity of the bacteria-produced human IL-10.
白细胞介素-10（IL-10）是一种强效抗炎细胞因子，在炎症性肠病中具有治疗应用。为了通过大肠杆菌作为载体底盘原位递送IL-10，设计了一种具有分泌生物活性IL-10能力的改良转运体。从头DNA合成包括一个561 bp的片段，该片段编码与T7启动子控制下的大肠杆菌密码子优化的成熟人IL-10基因框内融合的大肠杆菌外膜蛋白F的信号序列。该构建体在大肠杆菌实验室菌株大肠杆菌BL21（DE3）和大肠杆菌MDS42:T7中过表达。大肠杆菌BL21（DE3）周质和培养上清液中人类IL-10的平均浓度分别为355.8±86.3和5.7±1.7 ng/ml。重组大肠杆菌衍生的人IL-10的分子量为19 kDa，而在非还原条件下可以证明天然IL-10二聚体。脂多糖刺激的小鼠巨噬细胞中肿瘤坏死因子-α分泌的减少以及转录因子信号转导和转录激活蛋白3的激活形式的检测证明了细菌产生的人IL-10的生物活性。
