CD40突变体融合蛋白真核表达载体的构建及表达

目的:构建CD40突变体(muCD40)融合蛋白的真核表达载体pIRES2-EGFP/muCD40Ig,并在中国仓鼠卵巢细胞(CHO)中表达,以获得muCD40Ig融合蛋白,为检测体内可溶性muCD40分子建立实验基础。方法:从L929/muCD40基因转染细胞中通过RT-PCR扩增出人muCD40胞外段基因序列,从人脾脏细胞中扩增出人IgG1恒定区基因,分别插入真核表达载体pIRES2-EGFP,采用Superfectin转染CHO细胞,G418筛选出能稳定分泌muCD40Ig蛋白的基因转染细胞并亚克隆。筛选出的阳性克隆经无血清培养后,收集上清进行Western blot检测;并收集细胞进行RT-PCR鉴定,同时用流式细胞仪分析muCD40Ig蛋白与CD40L的结合能力。结果:成功构建了人pIRES2-EGFP/muCD40Ig重组真核表达载体,PCR及Western blot结果显示该CHO基因转染细胞能够稳定分泌人muCD40Ig蛋白;流式检测muCD40Ig融合蛋白能够与CD40L分子结合。结论:获得了能稳定分泌人muCD40Ig的CHO基因转染细胞株,muCD40Ig融合蛋白具有与CD40L结合的能力。

Construction of eukaryotic expression vector of muCD40-IgG1Fc fusion protein and its expression in CHO cells

AIM: To construct an eukaryotic expression vector of human muCD40Ig fusion protein,and to express it stably in Chinese hamster ovary(CHO) cells for obtaining muCD40Ig fusion protein and founding an experimental basis for investigating the soluble muCD40 molecule in vivo.METHODS: Extracellular domain of human muCD40 gene was amplified by RT-PCR from L929/muCD40-transfected cells,and the genes encoding the constant regions of human IgG1 were cloned from human splenocytes.The genes were inserted into eukaryotic expression vector pIRES2-EGFP,respectively.The recombinant vector was transfected into CHO cells by Superfectin.The transfected cells stably secreting muCD40Ig fusion protein was selected with G418 and subcloned.The serum-free culture supernatant of the selected positive clone was subjected to Western blotting and RT-PCR to confirm the expression of the fusion gene.The affinity of muCD40Ig and L929/CD40L was analyzed by flow cytometry(FCM).RESULTS: The eukaryotic expression vector pIRES2-EGFP/muCD40Ig was constructed successfully.PCR and Western blotting showed that the transfected CHO cell strain was able to secret muCD40Ig fusion protein stably.FCM demonstrated a good affinity between muCD40Ig and L929/CD40L.CONCLUSION: A transfected CHO cell strain stably expressing muCD40Ig fusion protein has been obtained,and the muCD40Ig fusion protein can bind to CD40L.