【摘要】 研制特异性鼠抗人PD-1功能性单克隆抗体,并对其生物学特性进行鉴定。以高表达人PD-1分子的基因转染细胞L929/PD-1作为免疫原,常规免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术进行细胞融合,以L929/PD-1作为抗体筛选细胞,L929/mock为对照细胞,经间接免疫荧光标记和流式细胞术分析、反复筛选和多次克隆化培养,筛选出分泌特异性鼠抗人PD-1单克隆抗体的杂交瘤细胞株;采用Western blot、Ig亚型快速定性试纸法、间接免疫荧光法、竞争结合抑制试验和MTT增殖实验对单抗进行生物学特性的分析。结果表明,成功获得1株特异、稳定分泌鼠抗人PD-1单克隆抗体的杂交瘤细胞株,命名为6E2。对6E2生物学功能的研究结果提示,该单抗能够识别活化T细胞表达的PD-1分子,且在体外能够显著抑制T细胞的增殖和细胞因子的分泌。获得的特异性鼠抗人PD-1功能性单克隆抗体,通过激发PD-1负性途径能够有效抑制T细胞的功能,为进一步研究PD-1信号奠定了物质基础。更多还原

【Abstract】 To prepare a functional mouse anti-human PD-1 monoclonal antibody and to characterize its biological activities,a stable human PD-1 transfected cell line L929/PD-1 was used as an antigen to immunize BALB/c mice.By means of the cell fusion technique,multiple cell subcloning,repeated screening with L929/PD-1 as target cells and the L929/mock cells used as the negative control,the hybridoma specifically secreting mouse anti-human PD-1 monoclonal antibody was generated.Then its biological characterization was investigated by Western blotting,rapid murine Ig-subclass typing,indirect immunofluorescene assay,mutual competitive inhibition test and MTT incorporation assay.The results of characterization study showed that the monoclonal antibody(named as 6E2) could bind to human PD-1 specially,and it could recognize a new epitope of PD-1.Furthermore,the mAb 6E2 could suppress T cells proliferation and cytokine secretion.It is evident that the functional monoclonal antibody for human PD-1 has been generated,and it would provide the initial material for further study on the role of human PD-1.更多还原