阻断CD40/CD40L相互作用延长移植物抗宿主病小鼠存活时间

目的：克隆表达人CD40-Ig融合蛋白，并在小鼠移植物抗宿主病（GVHD）模型中研究利用其阻断CD40/CD40L相互作用的保护性效果。方法：利用RT-PCR技术自人Daudi细胞系中克隆CD40基因胞外区，并插入含有人IgG1Fc段基因的pIG1载体中，构建携带CD40-Fc融合基因的瞬时表达载体转染COS-7细胞，间接夹心ELISA法检测CD40-Ig融合蛋白的表达，再将CD4-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，筛选分泌CD40-Ig融合蛋白的阳性重组CHO细胞并进行无血清大规模培养，收获培养上清利用ProteinA亲和层析法纯化融合蛋白。SDS-PAGE，Western blot和配基结合实验鉴定CD40-Ig的性质，将C57BL6/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CD40-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/40Ig和p3.1/40Ig。ELISA和Western blot确定在COS-7和CHO细胞中表达了CO40-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CD40-Ig可与CD40L结合，体内应用CD40-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：CD40/CD40L相互作用在GVHD的病理过程中可能扮演了十分重要的角色，提示人CD40-Ig融合蛋白在临床预防和治疗GVHD方面的巨大应用潜力。

Blockade of CD40/CD40L interactions prolongs the survival time of mice with graft-versus-host disease

AIM: To clone and express human CD40-Ig fusion protein, and further investigate its protective effects on graft-versus-host disease in a murine model. METHODS: Human CD40 gene extracellular region was cloned from Daudi cell line by RT-PCR and inserted into plasmid pIG1, which contains genomic human IgG1 Fc fragment. Then a transient vector containing CD40-Fc fusion gene was constructed and transfected into COS-7 cells. The secreted expression of CD40-Ig fusion protein was detected through indirect sandwich ELISA. In order to obtain plentiful CD40-Ig fusion protein, a constitutive vector was also generated by ligating the CD40-Fc fusion gene into pcDNA3.1, and hence transfecting it into CHO cells. Positive recombinant CHO cells, which secrete CD40-Ig fusion protein, were selected out and cultured on a large scale with serum-free medium, culture supernatant was harvested and 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 (iv) 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/40-Ig and p3.1/40-Ig were constructed as described above. Chimeric proteins were expressed in COS-7 cell and CHO cell culture supernatant and confirmed by ELISA and Western blot. SDS-PAGE showed that fusion protein has a disulfide-bonded dimeric structure and existed as homodimer. Purified CD40-Ig could bind to CD40L expressed on Jurkat cell line. In vivo administration of CD40-Ig could prevent the development of GVHD and significantly prolongs the mean survival time of mice with GVHD. CONCLUSION: The CD40/CD40L interactions play a pivotal role in the pathogenesis of GVHD, suggesting clinical potential for CD40-Ig in the prevention and treatment of human GVHD.