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目的 弄清只含白细胞介素 6 (IL 6 )受体 β亚基 (gp130 )胞外第二、三个FNⅢ区的可溶性突变体是否能拮抗IL 6的生物学效应。方法 首先用重叠延伸PCR方法扩增出gp130突变体cDNA ;接着将其克隆到真核表达载体pSVL中 ;用脂质体转染法将此表达载体导入CHO细胞中 ,通过点杂交和反转录PCR证明其得到有效表达 ;用MTT法在杂交瘤细胞和白血病细胞中观察了此突变体对IL 6生物学效应的影响。结果 发现此突变体的表达上清具有拮抗IL 6所致的杂交瘤细胞 7TD1增殖及白血病细胞R2生长抑制的作用。结论 gp130胞外第二、三个FNⅢ区中含有与IL 6 /IL 6Rα形成复合物的部位。此结果为深入研究gp130胞外区的结构与功能以及针对gp130胞外区的小分子激动剂和拮抗剂的设计提供了一些依据。
Objective To determine whether soluble variants containing only the extracellular second and third FNIII regions of the β subunit of interleukin 6 (ILβ) receptor (gp130) could antagonize the biological effects of IL 6. Methods The cDNA of gp130 mutant was amplified by overlap extension PCR. The recombinant plasmid was cloned into eukaryotic expression vector pSVL. The vector was transfected into CHO cells by lipofection. PCR was used to confirm the effect of this mutant on the biological effects of IL-6 in hybridoma cells and leukemia cells by MTT assay. The results showed that the supernatant of this mutant could inhibit the proliferation of 7TD1 cells induced by IL-6 and inhibit the growth of leukemia cells. Conclusions gp130 extracellular second, three FNIII contains IL6 / IL6Rα complex formation sites. This result provides some evidences for the further study of the structure and function of the extracellular domain of gp130 and the design of small molecule agonists and antagonists targeting the extracellular domain of gp130.