人CD40-Ig融合蛋白在CHO细胞中的表达与纯化研究

目的 建立稳定表达CD40－Ig融合蛋白的工程细胞株，获得大量融合蛋白以研究靶向阻断CD40：CD40L途径防治移植物抗宿主病（GVHD）策略的应用潜力。方法 自真核瞬时表达载体pIG／40Ig中切下CD40－Fc融合基因，插入pcDNA3．1载体中构建稳定表达载体。利用脂质体Lipo-fectamine将该载体转染CHO细胞，G418筛选抗性克隆；夹心ELISA法检测上清中CD40－Ig融合蛋白的表达；Westem blot法鉴定其免疫学活性。利用有限稀释法对筛选出的混合克隆单克隆化。滚瓶法无血清大批培养工程细胞，ProteinA亲和层析法纯化，SDS－PAGE后薄层扫描分析纯度。利用流式细胞术检测该蛋白与Jurkat细胞表面CD40L的结合功能。结果 CD40－Fc融合基因插入pcDNA3．1载体后构建成功稳定表达载体p3．1／40Ig，转染CHO细胞后经2次克隆化获得稳定表达CD40－Ig融合蛋白的基因工程细胞株，命名为p3．1／40Ig，转染CHO细胞后经2次克隆化获得稳定表达CD40－Ig融合蛋白的基因工程细胞株，命名为B2。ProteinA亲和层析纯化融合蛋白纯度达95％以上，该融合蛋白可特异结合Jukat细胞表面的CD40L。结论 CD40－Ig融合蛋白可模仿天然分子与其配基结合，为研究靶向CD40：CD40L途径的治疗制剂在防治GVHD中的潜在应用提供了有用的工具。

Study of expression and purification of CD40-Ig fusion protein in CHO cells

Objective To obtain CD40 Ig fusion protein, and investigate the therapeutic potentials of blocking the CD40/CD40L signal pathway in the prevention and treatment of graft versus host disease (GVHD). Methods The CD40 Fc fusion gene, derived from mammalian transient expressing vector pIG/40Ig, was inserted into constitutive expressing vector pcDNA3.1. Then the recombinant expression vector was transfected into CHO cells with Lipofectamine reagent. The CHO cells, secreting CD40 Ig fusion protein in the culture supernatant, were obtained after selecting by G418. That was confirmed by Sandwich ELISA and Western bolt assay. The transfected CHO cells were cloned two times by limited dilution method. After big batch serum free culture using roller bottle, the concentrated supernatant was purified by rProtein A affinity chromatograph method. The purity of the purified recombinant was confirmed by SDS PAGE. The binding of the CD40 Ig fusion protein to Jurkat cell was detected by FCAS. Results Mammalian constitutive expression vector p3.1/40Ig was constructed as described above. Then the mixed CHO clones, expressing CD40 Ig fusion protein, were selected by G418 0.8g/L for two weeks after transfection. Through twice limited dilution cloning, five single clones were obtained, and the best one named as B2. The big batch culture supernatant of B2 was harvested, concentrated, and purified through rProtein A affinity chromatograph. The purity of recombinant CD40 Ig protein is above 95%. FACS results shown that CD40 Ig can bind to the CD40L expressing on the Jurkat cells, which were stimulated by PHA P. Conclusion The CD40 Ig could mimic the native CD40 molecule and bind to its ligand. It provided a useful tool to investigate the role of blockade CD40/CD40L costimulation signal pathway in the prevention and therapy of GVHD.