抗人CD25人鼠嵌合抗体在CHO细胞中的稳定表达及初步鉴定

目的在CHO细胞中稳定表达抗人CD25人鼠嵌合抗体,并对抗体活性进行初步鉴定。方法采用脂质体法将嵌合抗体真核表达质粒pOptiVEC-H和pcDNA3.3-L共转染CHO-DHFR-细胞,通过去除HT和在培养基中加入500μg/ml的Geneticine进行阳性克隆的筛选,有限稀释法对阳性克隆进行亚克隆,通过在培养基中加入浓度逐步增加的MTX提高克隆的抗体表达量。采用流式细胞术(FCM)检测嵌合抗体的抗原结合活性及人抗体重链Fc段、轻链κ链;提取细胞基因组进行PCR鉴定;抗体经超滤浓缩后,采用蛋白G亲和纯化法进行纯化,并进行Westernblot分析;体外连续培养细胞株2个月及冻存、复苏后,采用ELISA法检测抗体分泌的稳定性。结果获得稳定分泌嵌合抗体的细胞株1C1,ELISA检测抗体表达量为103ng/ml;PCR结果表明,表达的质粒已整合入细胞基因组中;FCM及Westernblot结果显示,嵌合抗体含有人抗体重链Fc段及轻链κ链,且保留了鼠抗体V区的抗原结合特异性;经蛋白G亲和纯化后,获得抗体220μg,纯度>97%;体外连续培养细胞株2个月及冻存、复苏后,抗体分泌保持稳定。结论获得了稳定表达人CD25人鼠嵌合抗体的CHO细胞株,其具有鼠源抗体的抗原结合特异性及人抗体的恒定区。

Stable Expression of Human-Mouse Chimeric Antibody against Human CD25 in CHO Cells and Identification of Expressed Product

Objective To stably express the human-mouse chimeric antibody against human CD25 in CHO cells and prelimi-narily determine its activity. Methods CHO-DHFR-cells were co-transfected with eukaryotic expression vectors pOptiVEC-H and pcDNA3. 3-L for the chimeric antibody in mediation of liposome, and the positive clones were screened by removing HT and adding 500 μg / ml of Geneticine in the medium, then subcloned by limiting dilution. The MTX at gradually increased concentrations was added into the medium to enhance the antibody expression. The binding activity to antigen of chimeric antibody as well as the Fc fragment of heavy chain and κ chain of human antibody were determined by flow cytometry(FCM). The genome of cells was extract-ed for identification by PCR. The antibody was concentrated by ultrafiltration, purified by protein G affinity chromatography and ana-lyzed by Western blot. The antibody secreting stability was monitored by culturing the cells continuously for 2 months in vitro and by thawing the cells stored in frozen. Results The cell strain 1C1 stably secreting chimeric antibody was obtained, in which the antibody expression level determined by ELISA was 103 ng / ml. PCR results showed that the plasmid was integrated into the genome of cells. FCM and Western blot showed that the chimeric antibody contained human antibody Fc fragment and κ light chain, and retained the antigen binding specificity of mouse antibody V region. After purification by protein G affinity chromatography, 220 μg of antibody was obtained, with a purity of more than 97%. After the cells were cultured continuously in vitro for 2 months or the cells stored in frozen were thawed, the antibody was secreted stably. Conclusion A CHO cell strain stably expressing human-mouse chimeric antibody against human CD25 was obtained, which retained the antigen binding specificity of murine antibody and contained the constant region of human antibody.