摘要
【目的】制备生长激素受体的单克隆抗体,探讨其对生长激素受体的激活作用,为研究生长激素模拟物提供新思路。【方法】以生长激素受体胞外域(GHBP)免疫BALB/c鼠,通过杂交瘤技术制备抗生长激素受体的单克隆抗体,用流式技术筛选抗体,通过流式、激光共聚焦显微镜观察研究该单克隆抗体对生长激素受体的激活作用,然后通过Western-blot等技术手段研究其胞内的信号通路。【结果】通过杂交瘤技术共获得了30个分泌抗生长激素受体的单克隆抗体,经流式分析筛选到具有受体激动剂活性的单克隆抗体(命名为L8)。竞争性结合分析结果表明,L8在受体上的结合表位与生长激素重叠;激光共聚焦观察结果表明,L8不但能结合到细胞表面的生长激素受体,而且还能内化进入胞体。经Western-blot和流式分析表明,L8能与转染GHR基因的CHO-GHR638细胞表面的生长激素受体结合,并启动JAK2-STAT5、ERK1/2信号转导通路,且呈现出剂量依赖性。【结论】生长激素受体抗体可以作为受体的激动剂,其可能是通过诱导受体构象变化的方式激活受体,并启动JAK2-STAT5等信号转导通路。
【Objective】This study prepared monoclonal antibody of growth hormone receptor and investigated its effect on activation of growth hormone receptor to provide new ideas for studying growth hormone analog.【Method】The monoclonal growth hormone receptor antibody to GH was prepared through hybridoma technology using immunized BALB/c mice with extracellular domain（GHBP）.Then,monoclonal antibody was screened by flow cytometry.Its effect on activation of growth hormone receptor was also explored by flow cytometry and laser confocal microscope technology and the intracellular signaling pathways were explored by Western-blot.【Result】A total of 30 monoclonal antibodies secreting antigrowth hormone receptor were obtained by hybridoma technique,and monoclonal antibody with receptor agonist activity（named as L8）was screened by flow cytometry.Competitive binding analysis showed that L8 and growth hormone overlapped on epitope of receptor.Laser confocal microscope observation showed that L8 can not only combine to growth hormone receptor on cell surface,but also internalize into cell body.L8 can bind to growth hormone receptor on the surface of CHO-GHR638 which transfected GHR gene and trigger JAK2-STAT5 and ERK1/2signal transduction pathway with dose-dependence.【Conclusion】Antibodies of growth hormone receptor can be used as receptor agonist.It may activate receptor byinducing conformational changes of the receptor and triggering JAK2-STAT5 signal transduction pathway.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2016年第5期13-19,共7页
Journal of Northwest A&F University(Natural Science Edition)
基金
吉林省教育厅应用基础项目"猪生长激素模拟肽与生长激素受体结合特异性的研究"(吉教科合字[2012]第39号)
"十二五"农村领域国家科技计划项目(2013BAD10B00)
关键词
生长激素
抗生长激素受体抗体
信号转导通路
growth hormone
anti-growth hormone receptor antibody
signal transduction pathway
