人源性抗-D基因工程抗体在真核细胞CHO中的表达

Expression of Human Anti-Rhesus D Antibody in Eukaryotic CHO Cells

目的于真核细胞系CHO细胞中表达人源性抗-D基因工程抗体,为人源性抗-D基因工程抗体在临床上的应用打下坚实的基础。方法脂质体法将真核表达载体pAH4604/VH和pAG4622/VL共转染体外培养的CHO细胞,经霉酚酸及组氨醇筛选阳性克隆细胞。通过ELISA法检测培养上清中人IgG的表达量。应用化学发光WesternBlotting法对真核表达载体pAH4604/VH和pAG4622/VL在CHO细胞中的表达进行鉴定。结果pAH4604/VH和pAG4622/VL共转染体外培养的CHO细胞,经霉酚酸及组氨醇筛选12～14d后有阳性克隆长出,未转染细胞则全部死亡。筛选出的各阳性细胞克隆经扩大培养及ELISA定量检测其上清人IgG含量最高为4.05ng/ml。WesternBlotting显示上清和细胞内均有人IgG重链和轻链的表达。结论所构建的真核表达载体pAH4604/VH和pAG4622/VL在CHO真核细胞系表达成功,可产生人源化的IgG重链和轻链。

Objective To express human anti-Rhesus D antibody in eukaryotic CHO cells. Methods Recombinant eukaroytic expression vectors pAH4604/VH and pAG4622/VL were co-transfected into murine CHO cells using lipofectin. Positive clones were selected by including the selective agent mycophenolic acid （MPA）and histidinol in the culture medium. The quantity of human IgG in culture supernatant was determined by ELISA method. Expression of pAH4604/VH and pAG4622/VL was analyzed by chemiluminescence Western blot. Results Positive clones were obtained 14 days after the co-transfectlon and selection by mycophenolic acid（MPA）and histidinol. Nontransfected cells were all dead. The selected positive clones were expanded and the level of IgG in the culture supernatant was measured. The highest concentration of lgG detected in the supernatant was 4.05 ng/ml. Expression and production of both heavy chain and light chain of human IgG were confirmed by Western blotting analysis. Conclusion The recombinant eukaroytic expression vectors pAH4604/VH and pAG4622/VL can be successfully expressed in eukaryotic CHO cell line. The selected positive cells can produce human anti-Rhesus D IgG heavy chain and light chain.