抗人CD25人鼠嵌合抗体的构建、表达及其生物学活性的研究

发布时间：2018-05-28 10:33

本文选题：CD25 + 嵌合抗体　；参考：《武汉生物制品研究所》2010年博士论文

【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。
[Abstract]:The aim of this study was to modify the anti-human CD25 murine monoclonal antibody prepared by our department, and then to express it stably in CHO cells in order to obtain a cell line secreting stable and specific anti-human CD25 mouse chimeric antibody. The biological activity of the expressed chimeric antibody was identified to lay a solid foundation for the development of a more safe and effective therapeutic antibody and to make progress in the development of genetic engineering antibody. Firstly, the total RNAs of CD25 hybridoma cells were extracted. After reverse transcription into cDNA, several sets of degenerate primers for the variable region signal peptide of mouse antibody light chain and specific primers for the constant region of mouse antibody were designed. The variable region weight light chain gene of antibody was isolated by PCR method. The plasmid PAG4622 containing the constant region gene of human antibody was used as template to catch the constant region gene of weight and light chain of human antibody. The variable region of mouse antibody was spliced with the constant region gene of human antibody by PCR method. The spliced heavy light chain human mouse chimeric gene was ligated with the expression plasmid pOptiVEC and pcDNA3.3, respectively. The expression plasmid was cotransfected into 293T cells for transient expression using liposome method, and the antibody content in the supernatant was determined by double antibody sandwich ELISA method. Flow cytometry (FCM) was used to identify the antigen-binding activity of the expressed chimeric antibody. The results showed that the expressed chimeric antibody had the correct antigen-binding activity and contained the sequence of constant region of human antibody. The eukaryotic expression plasmid was successfully constructed. Then, CHO-dhfr_ cells were used as host cells, and the expression plasmid pOptiVEC-H and pcDNA3.3-L were cotransfected with liposome method to carry out the stable expression of antibody. Elisa quantitative results showed that the expression of antibody was 103 ng / ml at MTX 300nM, and purified and quantified by culture supernatant. The purity analysis of SDS-PAGE protein showed that the antibody purity was 97, and the antibody contained a complete anti-body weight light chain WB. The results showed that the chimeric antibody contained human antibody light chain constant region sequence, and WB showed that the purified antibody could be specific. Sex recognition of human CD25 molecule; The results of competitive analysis showed that chimeric antibodies recognized the same epitopes with parental antibodies, and there was a competitive relationship between chimeric antibodies and parental antibodies. The results showed that chimeric antibodies could inhibit the proliferation of T lymphocytes stimulated by PHA. The affinity constant of chimeric antibody determined by biosensor method was 1.9 脳 10 ~ (10) L / mol ~ (-1) L / mol ~ (-1) C _ 1 cell line. In conclusion, the eukaryotic expression plasmid of anti-human CD25 human mouse chimeric antibody was successfully constructed and stably expressed in CHO cells. The expressed antibody had specific antigen-binding activity and corresponding biological function. It lays the experimental foundation for the development of anti-human CD25 genetic engineering antibody.
【学位授予单位】：武汉生物制品研究所
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

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