CAJ | 학술논문

目的：建立新的白介素－6受体（ interleukin－6 receptor， IL－6R）小分子拮抗剂高通量筛选模型。方法PCR扩增IL－6R胞外区基因，将其克隆至真核表达载体构建重组质粒pABHis－IL6R，瞬时转染HEK293T细胞进行分泌表达。利用Western 印迹实验、受体配体结合实验对表达产物进行活性验证。利用二者的相互作用以及Fc片段与酶标二抗结合的特性建立基于ELISA法的IL－6R拮抗剂筛选模型，并用Z′－因子检测与已知拮抗剂ab47215对该模型的稳定性与可靠性进行评估。结果成功构建了真核表达载体pABHis－IL6R，并对IL－6R进行了分泌表达。所表达的IL－6R能够被特异性抗体识别，能够与其配体rhIL－6特异性地结合，并表现出良好的量效关系。经测算得出，该模型的Z′－因子为0．53，满足高通量筛选的要求。 ab47215能够剂量依赖性地阻断IL－6R与其配体的结合，其IC50＝（0．55±0．11）μg／ml。结论建立了一种新颖而简便的IL－6R拮抗剂筛选模型，为寻找高效、低毒的小分子拮抗剂奠定了基础。
목적：건립신적백개소－6수체（ interleukin－6 receptor， IL－6R）소분자길항제고통량사선모형。방법PCR확증IL－6R포외구기인，장기극륭지진핵표체재체구건중조질립pABHis－IL6R，순시전염HEK293T세포진행분비표체。이용Western 인적실험、수체배체결합실험대표체산물진행활성험증。이용이자적상호작용이급Fc편단여매표이항결합적특성건립기우ELISA법적IL－6R길항제사선모형，병용Z′－인자검측여이지길항제ab47215대해모형적은정성여가고성진행평고。결과성공구건료진핵표체재체pABHis－IL6R，병대IL－6R진행료분비표체。소표체적IL－6R능구피특이성항체식별，능구여기배체rhIL－6특이성지결합，병표현출량호적량효관계。경측산득출，해모형적Z′－인자위0．53，만족고통량사선적요구。 ab47215능구제량의뢰성지조단IL－6R여기배체적결합，기IC50＝（0．55±0．11）μg／ml。결론건립료일충신영이간편적IL－6R길항제사선모형，위심조고효、저독적소분자길항제전정료기출。
Objective To establish a high throughput screening assay for identifying human small molecular antagonists targeted IL-6R.Methods The full length gene of the human IL-6R extracellular region was amplified by PCR and cloned into a eukaryotic expression vector to construct recombination expression plasmid pABHis -IL6R that was then transfected transiently into HEK293T cells to prepare recombination protein IL-6R.Western blotting assay and receptor-ligand binding experiment were used to analyze the bioactivity of IL-6R.A new screening method based on ELISA was established using the function of IL-6R binding to its ligand and the characteristics of Fc fragment binding to IgG-HRP.Then Z′-factor was calculated and a known antagonist ab 47215 was used to assess the stability and reliability of the new assay .Results Recombination plasmid pABHis-IL6R was constructed and soluble IL-6R was prepared.IL-6R reported herein could be recognized by an anti-IL-6R antibody and specifically bind to its ligand in a dose response manner .A Z′-factor of 0.53 was obtained that could serve high throughput screening assay .Ab47215 , as a known specific antagonist , was able to block rhIL-6 from binding to the receptor in a dose-dependent manner in the new screening assay , the IC50 of which was (0.55 ± 0.11)μg/ml.Conclusion An innovative and easy screening assay for identifying human IL-6R antagonists is established , which might help discover potent and specific antagonists .