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

I基因合成了白介素10(IL10)和粒细胞集落刺激因子(GCSF)的融合蛋白，白介素10促进激活的中性粒细胞凋亡，粒细胞集落刺激因子与中性粒细胞高度结合，在血液中保留较长的半衰期。用RT-PCR法将人GCSFc DNA和人IL10c DNA亚克隆到pSP73载体上，作为感受态细胞在DH5α中扩增。我们设计了野生型IL10、野生型IL10(IL10M)、野生型GCSF与IL10M融合蛋白以及拮抗型GCSF与IL10M融合蛋白，并利用无细胞蛋白质合成系统进行翻译。用抗IL10和GCSF抗体的夹心ELISA法检测到合成的融合蛋白上的这两个表位。使用抗IL10抗体的流式细胞术显示融合蛋白与人白细胞结合。野生型IL-10和IL-10M能强烈抑制脂多糖刺激的白细胞产生炎性细胞因子IL-12和肿瘤坏死因子α，其抑制程度与IL-10相当。这些结果有力地表明，合成的IL10M和GCSF融合蛋白有望成为新型抗炎药物的候选药物。

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.