gp130胞外区两个片段的克隆与表达及其对IL-6信号转导的影响(英文)

Cloning and Expression of Two Fragments of gp130 Extracellular Domain and Their Effects on IL-6 Signal Transduction

gp130是白细胞介素6(IL-6)受体的β亚基和信号转导子。IL-6介导的信号转导起始于gp130与IL-6和IL-6Rα形成的复合体,但目前还不了解gp130分子中参与复合物形成的部位。为了研究gp130胞外区的结构与功能,我们通过PCR方法构建并克隆表达了其胞外区全长(大片段)及氨基端304个氨基酸残基的片段(小片段),并在杂交瘤细胞系7TD1和肝癌细胞系HepG2中观察其生物学效应。结果发现,含细胞因子结合区的可溶性小片段和大片段都能与膜结合型gp130竞争性结合IL-6和IL-6Rα,拮抗IL-6信号。可见gp130氨基端304个氨基酸残基的片段中存在与IL-6和IL-6Rα作用的部位。

Gpl30 is the β subunit and signal transducer of interleukin-6 receptor(IL-6R). Signalling of IL-6 is initiated by the complex formation of gp130 with IL-6 bound to IL-6Rα, but we know nothing about the part of gp130 which does take part in the complex formation. Therefore, we have firstly made two soluble gp130 cDNA (the extracellular domain and the N-terminal part including amino acids 1 - 304), then cloned them into the expression vector pSVL and expressed them in CHO cells. The expression of these soluble gp130 was detected by in situ hybridization and Western blot methods. We studied their biological effects on IL-6 stimulated cells and observed that both soluble gp130 proteins inhibited IL-6 induced proliferation of a hybridoma cell line 7TD1 and the expression of acute phase protein (APP) gene promoters connected report genes and the binding activities of nuclear factor APRF to double-strand oligonucleatides probes in a hepatoma cell line HepG2. These results suggested that both soluble gp130 molecules antagonized the activities of IL-6. The binding sites of gp130 to IL-6 and IL-6Rα maybe exist in the N-terminal 1 - 304 amino acids of gp130 extracellular domain.