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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?
Objective To investigate the protective effect of blocking CD4 0 / CD4 0L interaction with human CD4 0 Ig in mouse GVHD model. Methods The extracellular domain of human CD4 0 gene was inserted into the pIG1 vector containing the human IgG1 Fc gene. The transient expression vector carrying CD4 Fc fusion gene was constructed and transfected into COS7 cells. The expression of CD4 0 Ig fusion protein was detected by ELISA. Then the CD4 0 Fc fusion gene was inserted into the corresponding site of pcDNA3 1 to construct a stable expression vector for transfection into CHO cells and the fusion protein was purified by Protein A affinity chromatography. SDS PAGE, Western blot and ligand binding assay to determine the properties of CD4 0 Ig. Splenocytes of C5 7BL6 / J (H2b) mice were injected via tail vein into sublethally irradiated BALB / c (H2d) mice to establish an acute GVHD model and evaluated by in vivo injection of CD4O Ig fusion protein Its protective effect on acute GVHD mice. Results The mammalian expression vectors pIG / 40Ig and p3I / 40Ig were constructed as described above. ELISA and Western blot confirmed that CD4 0 Ig fusion protein was expressed in COS7 and CHO cells. SDS PAGE results show that the protein has a disulfide-bonded dimeric structure and exists as a homodimer. Purified CD4O Ig can bind to CD4OL. Treatment with CD4 0 Ig fusion protein in vivo delayed the progression of GVHD in mice and significantly prolonged the mean survival time in mice. Conclusions Our results demonstrate that the CD4 0 / CD4 0L interaction may play a very important role in the pathogenesis of GVHD and suggest the great utility of human CD4 0 Ig fusion protein in the clinical prevention and treatment of GVHD.