摘要
目的 在小鼠GVHD模型中研究利用人CD4 0 Ig阻断CD4 0 /CD4 0L相互作用的保护性效果。方法 将人CD4 0基因胞外区插入含有人IgG1Fc段基因的pIG1载体中 ,构建携带CD4 0 Fc融合基因的瞬时表达载体转染COS 7细胞 ,ELISA法检测CD4 0 Ig融合蛋白的表达。再将CD4 0 Fc融合基因连接入pcDNA3 1的相应位点构建稳定表达载体转染CHO细胞 ,利用ProteinA亲和层析法纯化融合蛋白。SDS PAGE、Westernblot和配基结合实验鉴定CD4 0 Ig的性质。将C5 7BL6 /J(H 2 b)小鼠的脾细胞经尾静脉注射入亚致死剂量照射的BALB/c(H 2 d)小鼠体内建立急性GVHD模型 ,通过体内注射CD4 0 Ig融合蛋白评价其对急性GVHD小鼠的保护效果。结果 按上述方法分别构建了哺乳动物表达载体pIG/ 4 0Ig和p3 1/ 4 0Ig。ELISA和Westernblot确定在COS 7和CHO细胞中表达了CD4 0 Ig融合蛋白。SDS PAGE结果显示该蛋白具有通过二硫键结合的二聚体结构并以同源二聚体的形式存在。纯化的CD4 0 Ig可与CD4 0L结合。体内应用CD4 0 Ig融合蛋白治疗可延缓小鼠GVHD病情发展并显著延长小鼠的平均存活时间。结论 我们的结果证明CD4 0 /CD4 0L相互作用在GVHD的病理过程中可能起到了十分重要的作用 ,提示人CD4 0 Ig融合蛋白在临床预防和治疗GVHD方面的巨大应用?
Abstract:Objective To investigate the protective effects of blocking CD40/CD40L interactions with human CD40-Ig fusion protein in a murine graft-versus-host disease model.Methods Human CD40 gene extracellular region was inserted into plasmid pIG1, which contains genomic human IgG1 Fc gene. A transient vector containing CD40-Fc fusion gene was transfected into COS-7 cells. The CD40-Ig fusion protein was detected through enzyme-linked immunosorbent assay (ELISA). A constitutive vector was also generated by ligating the CD40-Fc fusion gene into pcDNA3.1 and transfecting it into CHO cells. CD40-Ig was purified by protein A affinity chromatography. SDS-PAGE, Western blot and ligand binding assay were used to identify the qualities of CD40-Ig. Murine acute graft-versus-host disease (GVHD) was induced by intravenous injection of C57BL/6J (H-2b) spleen cells into sub-lethally irradiated BALB/c (H-2d) mice. Protective effects against murine graft-versus-host disease by in vivo administration of CD40-Ig were evaluated.Results Mammalian expression vectors pIG/40Ig and p3.1/40Ig were constructed as described above. Chimeric proteins were expressed in COS-7 and CHO cell culture supernatant and confirmed by ELISA and Western blot. SDS-PAGE showed that fusion proteins had a disulfide-bonded dimeric structure and existed as homodimer. Purified CD40-Ig could bind to CD40L. In vivo administration of CD40-Ig could prevent the development of GVHD and significantly prolong the mean survival time of mice with graft-versus-host disease.Conclusions These results demonstrate that CD40/CD40L interactions play an important role in the pathogenesis of graft-versus-host disease and suggest clinical potential for CD40-Ig in the prevention and treatment of human graft-versus-host disease.
