本文選題：CD25 + 嵌合抗體��； 參考：《武漢生物制品研究所》2010年博士論文

【摘要】： 本課題旨在對本科室自行研制的抗人CD25鼠源單抗進行人鼠嵌合改造,然后在CHO細胞中進行穩(wěn)定表達,以期獲得穩(wěn)定分泌特異性的抗人CD25人鼠嵌合抗體的細胞株,并對表達的嵌合抗體的生物學活性進行鑒定,為開發(fā)更加安全有效的治療性抗體打下物質(zhì)基礎,同時也期在基因工程抗體研制方面取得進展。 首先提取CD25雜交瘤細胞總RNA,逆轉(zhuǎn)錄成cDNA后,設計多組針對鼠抗體重輕鏈可變區(qū)信號肽的簡并引物及針對鼠抗體恒定區(qū)的特異性引物,通過PCR法釣取抗體可變區(qū)重輕鏈基因。以含人抗體恒定區(qū)基因的質(zhì)粒PAG4622為模板釣取人抗體重輕鏈恒定區(qū)基因,測序鑒定正確后通過PCR法將鼠抗體可變區(qū)與人抗體恒定區(qū)基因進行拼接。將拼接后的重輕鏈人鼠嵌合基因與表達質(zhì)粒pOptiVEC和pcDNA3.3分別進行連接。使用脂質(zhì)體法將表達質(zhì)粒共轉(zhuǎn)染293T細胞進行抗體的瞬時表達;使用雙抗夾心ELISA法對細胞上清中抗體進行含量測定;使用流式細胞術(FCM)對表達的嵌合抗體的抗原結(jié)合活性進行鑒定。結(jié)果表明,表達的嵌合抗體具有正確的抗原結(jié)合活性,同時含有人抗體恒定區(qū)序列,真核表達質(zhì)粒構(gòu)建成功。 然后以CHO-dhfr_細胞作為宿主細胞,采用脂質(zhì)體法共轉(zhuǎn)染表達質(zhì)粒pOptiVEC-H和pcDNA3.3-L進行抗體的穩(wěn)定表達。ELISA定量結(jié)果顯示,在MTX 300nM時抗體表達量為103ng/ml;通過對培養(yǎng)上清純化、定量,一共收獲抗體220μg;SDS-PAGE蛋白純度分析表明抗體純度97%,同時含有完整的抗體重輕鏈;WB結(jié)果顯示嵌合抗體含有人抗體重輕鏈恒定區(qū)序列;Dot Blotting及WB結(jié)果顯示純化的抗體能特異性識別人CD25分子;競爭性分析結(jié)果表明嵌合抗體與親本抗體之間識別相同表位,存在競爭關系;CCK結(jié)果顯示,嵌合抗體對PHA刺激的T淋巴細胞增殖存在抑制作用;生物傳感器法測得嵌合抗體的親和常數(shù)為1.9×1010L/mol;1C1細胞株體外連續(xù)培養(yǎng)3個月抗體分泌保持穩(wěn)定。 綜上所述,本試驗成功構(gòu)建了抗人CD25人鼠嵌合抗體真核表達質(zhì)粒,并在CHO細胞中穩(wěn)定表達,表達的抗體具有特異抗原結(jié)合活性及相應生物學功能,為抗人CD25基因工程抗體的開發(fā)奠定了試驗基礎。
[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.
【學位授予單位】：武漢生物制品研究所
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R392

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