【作者】 潘勇兵；

【导师】 杨晓明；

【作者基本信息】 武汉生物制品研究所 ， 免疫学， 2010， 博士

【摘要】 本课题旨在对本科室自行研制的抗人CD25鼠源单抗进行人鼠嵌合改造,然后在CHO细胞中进行稳定表达,以期获得稳定分泌特异性的抗人CD25人鼠嵌合抗体的细胞株,并对表达的嵌合抗体的生物学活性进行鉴定,为开发更加安全有效的治疗性抗体打下物质基础,同时也期在基因工程抗体研制方面取得进展。首先提取CD25杂交瘤细胞总RNA,逆转录成cDNA后,设计多组针对鼠抗体重轻链可变区信号肽的简并引物及针对鼠抗体恒定区的特异性引物,通过PCR法钓取抗体可变区重轻链基因。以含人抗体恒定区基因的质粒PAG4622为模板钓取人抗体重轻链恒定区基因,测序鉴定正确后通过PCR法将鼠抗体可变区与人抗体恒定区基因进行拼接。将拼接后的重轻链人鼠嵌合基因与表达质粒pOptiVEC和pcDNA3.3分别进行连接。使用脂质体法将表达质粒共转染293T细胞进行抗体的瞬时表达;使用双抗夹心ELISA法对细胞上清中抗体进行含量测定;使用流式细胞术(FCM)对表达的嵌合抗体的抗原结合活性进行鉴定。结果表明,表达的嵌合抗体具有正确的抗原结合活性,同时含有人抗体恒定区序列,真核表达质粒构建成功。然后以CHO-dhfr_细胞作为宿主细胞,采用脂质体法共转染表达质粒pOptiVEC-H和pcDNA3.3-L进行抗体的稳定表达。ELISA定量结果显示,在MTX 300nM时抗体表达量为103ng/ml;通过对培养上清纯化、定量,一共收获抗体220μg;SDS-PAGE蛋白纯度分析表明抗体纯度>97%,同时含有完整的抗体重轻链;WB结果显示嵌合抗体含有人抗体重轻链恒定区序列;Dot Blotting及WB结果显示纯化的抗体能特异性识别人CD25分子;竞争性分析结果表明嵌合抗体与亲本抗体之间识别相同表位,存在竞争关系;CCK结果显示,嵌合抗体对PHA刺激的T淋巴细胞增殖存在抑制作用;生物传感器法测得嵌合抗体的亲和常数为1.9×1010L/mol;1C1细胞株体外连续培养3个月抗体分泌保持稳定。综上所述,本试验成功构建了抗人CD25人鼠嵌合抗体真核表达质粒,并在CHO细胞中稳定表达,表达的抗体具有特异抗原结合活性及相应生物学功能,为抗人CD25基因工程抗体的开发奠定了试验基础。更多还原

【Abstract】 Basing on the anti-human CD25 hybridoma cell established by our laboratory, we constructed a human-mouse chimeric antibody expression plasmid against human CD25 and then expressed it in CHO cell. The aim was to establish a CHO cell line which stably expressed chimeric antibody against human CD25 and characterize its biological activity and lay foundation for the further development of safe and effective therapeutic antibody, and we also hoped to achieve progress in the study of genetically engineered antibody.Firstly, total RNA was extracted from the murine anti-human CD25 hybridoma cell line, the VH and VL gene of the hybridoma cells were amplified by RT-PCR with the degenerate primers binding to signal peptide sequences of the heavy and light variable chain and primers binding to constant region sequences. Human antibody heavy and light constant region were cloned by PCR using plasmid PAG4622 as a template. The mouse variable region and human constant region were spliced by PCR method, the heavy and light human-mouse chimeric genes were ligated with plasmids pOptiVEC and pcDNA3.3 respectively and cotransfected 293T cells throμgh lipofectomine method.The antibody expression lever was determined by sandwich ELISA; the antigen binding activity of the chimeric antibody was characterized by FCM. The results showed that the chimeric antibody could bind to the antigen and had human antibody constant region. The eukaryotic expression plasmids of chimeric antibody were constructed succesffuly.Secondly, plasmids pOptiVEC-H and pcDNA3.3-L were cotransfected CHO-dhfr- cells for stable expression.Quantitative ELISA result showed that the antibody expression lever was 103ng/ml when MTX concertration was 300nM;220μg antibody was harvested in total; the antibody purity was more than 97% analysed by SDS-PAGE; Western blotting result showed that the chimeric antibody had human heavy and light chain constant region; Dot Blotting and Western blotting results showed that the chimeric antibody could bind to CD25 molecule specifically; the competitive analysis showed that there was a competitive relationship between the chimeric and parental antibody and they may recognize the same epitope; CCK result showed that T lymphocytes proliferation could be inhibited by the chimeric antibody; biocore results showed that the affinity of chimeric antibody was about 1.9×1010l/mol; the cell line 1C1 could stably secret antibody for 3 months continuously.In summary, we constructed and expressed a human-mouse chimeric antibody against human CD25 in CHO cells successfully, which laid the foundation for the development of genetically engineered antibody against human CD25.更多还原