嵌合性抗ROR1抗体Fab片段的制备及鉴定  

Preparation and identification of chimeric anti-ROR1 antibody fab fragment

作　　者：殷郑娜 梅思伟 蒋秀敏 张宁芝[1] YIN Zheng-Na;MEI Si-Wei;JIANG Xiu-Min;ZHANG Ning-Zhi(Fuyang People′s Hospital,Fuyang 236000,China)

机构地区：[1]阜阳市人民医院,阜阳236000

出　　处：《中国免疫学杂志》2020年第13期1610-1614,共5页Chinese Journal of Immunology

摘　　要：目的:构建嵌合性抗ROR1抗体Fab片段原核表达载体,初步探讨该抗体的生物学特性。方法:利用小鼠免疫和细胞融合,选取阳性克隆细胞株,在此基础上,运用基因工程技术扩增得到抗体轻链和重链,经酶切后与原核表达质粒pETDUET-1连接,构建重组表达质粒,将该重组质粒转入E.coli BL21中表达、纯化,SDS-PAGE、Western blot及ELISA对该抗体进行鉴定和分析,流式细胞术及免疫荧光初步检测该抗体与卵巢肿瘤细胞的特异性结合能力。结果:筛选出分泌抗ROR1抗体的杂交瘤单克隆细胞株,在此基础上,成功扩增抗体轻链和重链并构建嵌合性抗ROR1抗体Fab段原核表达载体,经检测证实抗ROR1抗体Fab段可与ROR1抗原和卵巢肿瘤细胞特异性结合。结论:初步验证嵌合性抗ROR1抗体Fab片段可与卵巢肿瘤细胞结合,为进一步探究抗体肿瘤靶向治疗奠定了基础。Objective:A chimeric anti-ROR1 antibody Fab fragment prokaryotic expression vector was constructed to explore its biological characteristics.Methods:Mouse immunity and cell fusion technologies were used to select positive monoclonal cells.On this basis,genetic engineering techniques were used to amplify the light and heavy chains.After endonuclease digestion treatment,the light and heavy chains were connected to the prokaryotic expression plasmid pETDUET-1 to construct the recombinant expression plasmid,then which was transferred into E.coli BL21 to obtain anti-ROR1 antibody Fab fragment.SDS-PAGE,Western blot and ELISA were used to identify and analyze the anti-ROR1 antibody Fab fragment.Specific binding capacity of this antibody to ovarian tumor cells was initially examined by cytometry and immunofluorescence.Results:A hybridoma monoclonal cell secreting an anti-ROR1 antibody was selected,and on the basis of this,the antibody light chain and heavy chain were successfully amplified and then to construct a chimeric anti-ROR1 antibody Fab segment prokaryotic expression vector.Test results demonstrated that the anti-RORl antibody Fab fragment specifically binds to RORl antigen and ovarian tumor cells.Conclusion:Preliminary verification of the chimeric anti-ROR1 antibody Fab fragment can be combined with ovarian tumor cells,laying the foundation for further exploration of antibody-targeted tumor therapy.

关 键 词：ROR1 FAB片段 嵌合性抗ROR1抗体 卵巢癌 

分 类 号：R318[医药卫生—生物医学工程]