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Induction of activated leukocyte-selective apoptosis by the fusion protein IL10-GCSF

融合蛋白 IL10-GCSF 诱导激活的白细胞选择性凋亡

基本信息

批准号：
21591651
负责人：
HAYAMIZU Keisuke
金额：
$ 2.83万
依托单位：
Hiroshima International University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2009
资助国家：
日本
起止时间：
2009 至 2011
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-21591651/
关键词：
サイトカイン融合蛋白アポトーシス炎症好中球 GCSF IL10 DIC

项目摘要

I genetically synthesized the fusion proteins of interleukin 10(IL10) which facilitates apoptosis of the activated neutrophils and granulocyte colony-stimulating factor(GCSF) which highly binds neutrophils and is retained with longer half-lives in blood. cDNA of a human GCSF clone and that of human IL10 prepared from a volunteer by RT-PCR were subcloned into the plasmid pSP73 and propagated in DH5α as competent cells. The cDNA of antagonistic GCSF was synthesized by mutation at the site III, and that of IL10 was monomerized by elongation of the hinge between the helix D and E. I designed cDNAs of such wild-type IL10, monomerized IL10(IL10M), a fusion protein of wild-type GCSF and IL10M, and a fusion protein of antagonistic GCSF and IL10M, then translated them using a cell-free protein synthesis system. Sandwich ELISA with antibodies to IL10 and GCSF detected both epitopes on those synthesized fusion proteins. Flow cytometry using an anti-IL10 antibody demonstrated binding of the fusion proteins to human leukocytes. The wild-type IL10 and IL10M strongly suppressed production of inflammatory cytokines as IL12 and TNFα by LPS-stimulated leukocytes, and the degree of IL12 suppression by the fusion proteins was comparable to the IL10s. These results strongly suggest that the synthesized fusion proteins of IL10M and GCSF are promising candidates for new anti-inflammatory drugs.

我基因合成了促进活化的中性粒细胞凋亡的白细胞介素10（IL10）和粒细胞集落刺激因子（GCSF）的融合蛋白，后者与中性粒细胞高度结合，并在血液中保留较长的半衰期。将人GCSF克隆体cDNA和志愿者il - 10克隆体cDNA亚克隆到pSP73质粒中，并在DH5α中作为感受态细胞增殖。通过在III位点突变合成拮抗GCSF的cDNA，并通过螺旋D和e之间的铰链伸长将IL10的cDNA单体化。我设计了野生型IL10、单体化IL10（IL10M）、野生型GCSF与IL10M的融合蛋白、拮抗GCSF与IL10M的融合蛋白的cDNA，然后使用无细胞蛋白合成系统进行翻译。结合IL10和GCSF抗体的夹心ELISA检测了合成的融合蛋白上的两个表位。使用抗il - 10抗体的流式细胞术显示融合蛋白与人白细胞结合。野生型IL10和IL10M强烈抑制lps刺激的白细胞产生炎性细胞因子il - 12和tnf - α，融合蛋白对il - 12的抑制程度与IL10相当。这些结果强烈提示，合成的IL10M和GCSF融合蛋白是新型抗炎药物的有希望的候选者。