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共刺激分子配体PD-L1、PD-L2的真核表达及其转基因小鼠的制备

发布时间:2019-05-23 01:26
【摘要】:PD-L1、PD-L2是新发现的B7家族共刺激分子配体,二者具有共同的受体PD-1,并通过与受体PD-1结合抑制T细胞的增殖和过度活化,在细胞免疫应答过程中起负调控作用,同时通过影响细胞因子的分泌对体液免疫也发挥了调控作用。PD-L1、PD-L2/PD-1路径在机体自身免疫耐受和肿瘤细胞逃逸机体免疫监控过程中都发挥着重要作用。 本研究通过RT-PCR的方法成功地从PHA刺激的小鼠外周血单个核细胞和小鼠胸腺组织中克隆了PD-L1和PD-L2基因,并构建了重组真核表达载体pEF6/PD-L1-V5 His和pEF6/PD-L2-V5 His。两个重组质粒转染CHO细胞,经间接免疫荧光实验证明了构建的重组质粒可以CHO细胞中的成功表达PD-L1和PD-L2基因。稳定筛选后,经western-blot检测得到了稳定表达PD-L1和PD-L2的细胞株,为体外研究PD-L1和PD-L2对免疫调控的作用建立了细胞工具。 重组质粒经酶切,显微注射和PCR筛选分别得到了PD-L1的2只Found小鼠和PD-L2的1只Found小鼠,但只有PD-L2Found小鼠可通过生殖器官稳定遗传了PD-L2基因。并且通过southern-blot得到验证。 本研究通过克隆PD-L1和PD-L2基因来构建稳定表达细胞系和转基因小鼠是成功的。将为进一步研究PD-L1和PD-L2对免疫调控的作用提供有用的工具。
[Abstract]:PD-L1,PD-L2 is a newly discovered B7 family costimulatory molecule ligand, which share the same receptor PD-1, and inhibit the proliferation and overactivation of T cells by binding to the receptor PD-1. It plays a negative role in the process of cellular immune response, and also plays a regulatory role in humoral immunity by affecting the secretion of cytokines. PD-L1, PD-L2/PD-1 pathway plays an important role in autoimmune tolerance and immune monitoring of tumor cells. In this study, PD-L1 and PD-L2 genes were successfully cloned from mouse peripheral blood mononuclear cells and mouse thymic tissues stimulated by PHA by RT-PCR. The recombinant eukaryotic expression vectors pEF6/PD-L1-V5 His and pEF6/PD-L2-V5 His. were constructed. Two recombinant plasmids were transferred into CHO cells. Indirect immunofluorescence assay showed that the recombinant plasmid could successfully express PD-L1 and PD-L2 genes in CHO cells. After stable screening, the cell lines stably expressing PD-L1 and PD-L2 were detected by western-blot, which established a cell tool for studying the effect of PD-L1 and PD-L2 on immune regulation in vitro. Two Found mice of PD-L1 and one Found mouse of PD-L2 were obtained by restriction enzyme digestion, microinjection and PCR screening, respectively, but only PD-L2Found mice could stably inherit PD-L2 gene through reproductive organs. And verified by southern-blot. In this study, PD-L1 and PD-L2 genes were cloned to construct stable expression cell lines and transgenic mice. It will provide a useful tool for further study of the role of PD-L1 and PD-L2 in immune regulation.
【学位授予单位】:扬州大学
【学位级别】:硕士
【学位授予年份】:2005
【分类号】:R392.1

【参考文献】

相关期刊论文 前1条

1 徐宽枫;B7家族新成员PD-L1、PD-L2的研究进展[J];细胞与分子免疫学杂志;2003年05期



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