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

我基因合成的融合蛋白白细胞介素10（IL 10），促进活化的中性粒细胞和粒细胞集落刺激因子（GCSF），高度结合中性粒细胞，并保留在血液中具有较长的半衰期的细胞凋亡。用RT-PCR法从志愿者中制备人GCSF和人IL 10的cDNA克隆，将其亚克隆到质粒pSP 73中，并在DH 5 α中作为感受态细胞增殖。拮抗性GCSF的cDNA通过位点III的突变而合成，IL 10的cDNA通过螺旋D和E之间的铰链的延长而单体化。我设计了野生型IL 10、单体化IL 10（IL 10 M）、野生型GCSF和IL 10 M的融合蛋白以及拮抗性GCSF和IL 10 M的融合蛋白的cDNA，然后使用无细胞蛋白质合成系统翻译它们。用IL 10和GCSF抗体的夹心ELISA检测这些合成的融合蛋白上的两个表位。使用抗IL-10抗体的流式细胞术证明了融合蛋白与人白细胞的结合。野生型IL 10和IL 10 M强烈抑制LPS刺激的白细胞产生炎性细胞因子如IL 12和TNFα，融合蛋白对IL 12的抑制程度与IL 10相当。这些结果有力地表明，合成的IL 10 M和GCSF的融合蛋白是新的抗炎药物的有希望的候选者。

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.