Shantou University Medical College, Shantou, China.
Int Immunopharmacol. 2011 Jun;11(6):646-51. doi: 10.1016/j.intimp.2011.01.005. Epub 2011 Jan 19.
High-mobility group box-1 (HMGB1) plays important roles in inflammation, immune responses, and tumor progression. Since HMGB1 and its components have been shown to be mediators of a number of diseases but several sources of recombinant HMGB1 showed controversial biological activity, it is important to obtain recombinant HMGB1 with properties that resemble the native protein. For this purpose, we cloned genes coding for human HMGB1 and its active components A box and B box by PCR and inserted the cloned genes into pET28a vectors for transformation of Escherichia coli BL21. The E. coli expressed proteins were then purified with a Ni(2+)-NTA column and the endotoxin content was removed. Recombinant human HMGB1 (rhHMGB1) and its B box thus obtained stimulated, but A box inhibited, the production of the chemokine CXCL8/IL-8 by THP-1 monocytic cell line. We also used purified rhHMGB1 to immunize rabbits and generated potent anti-sera, which was capable of neutralizing the activity of rhHMGB1 in vitro and detecting the increased HMGB1 expression in inflammatory tissues in mice and humans. Thus, we have established essential means to produce biologically active rhHMGB1 that will facilitate us to study its role in diseases and to explore its potential as a therapeutic agent.
高迁移率族蛋白 B1（HMGB1）在炎症、免疫反应和肿瘤进展中发挥重要作用。由于 HMGB1 及其成分已被证明是许多疾病的介质，但几种来源的重组 HMGB1 表现出有争议的生物学活性，因此获得具有类似于天然蛋白特性的重组 HMGB1 非常重要。为此，我们通过 PCR 克隆了编码人 HMGB1 及其活性成分 A 盒和 B 盒的基因，并将克隆的基因插入 pET28a 载体中，用于转化大肠杆菌 BL21。然后，用 Ni(2+)-NTA 柱纯化大肠杆菌表达的蛋白质，并去除内毒素。由此获得的重组人 HMGB1（rhHMGB1）及其 B 盒刺激了 THP-1 单核细胞系产生趋化因子 CXCL8/IL-8，但 A 盒抑制了其产生。我们还使用纯化的 rhHMGB1 免疫兔子，产生了有效的抗血清，该抗血清能够在体外中和 rhHMGB1 的活性，并检测到在小鼠和人类炎症组织中 HMGB1 表达增加。因此，我们已经建立了生产具有生物学活性的 rhHMGB1 的必要手段，这将有助于我们研究其在疾病中的作用，并探索其作为治疗剂的潜力。
