共刺激分子配體PD-L1、PD-L2的真核表達及其轉基因小鼠的制備
發(fā)布時間：2019-05-23 01:26
【摘要】：PD-L1、PD-L2是新發(fā)現的B7家族共刺激分子配體,二者具有共同的受體PD-1,并通過與受體PD-1結合抑制T細胞的增殖和過度活化,在細胞免疫應答過程中起負調控作用,同時通過影響細胞因子的分泌對體液免疫也發(fā)揮了調控作用。PD-L1、PD-L2/PD-1路徑在機體自身免疫耐受和腫瘤細胞逃逸機體免疫監(jiān)控過程中都發(fā)揮著重要作用。 本研究通過RT-PCR的方法成功地從PHA刺激的小鼠外周血單個核細胞和小鼠胸腺組織中克隆了PD-L1和PD-L2基因,并構建了重組真核表達載體pEF6/PD-L1-V5 His和pEF6/PD-L2-V5 His。兩個重組質粒轉染CHO細胞,經間接免疫熒光實驗證明了構建的重組質�？梢訡HO細胞中的成功表達PD-L1和PD-L2基因。穩(wěn)定篩選后,經western-blot檢測得到了穩(wěn)定表達PD-L1和PD-L2的細胞株,為體外研究PD-L1和PD-L2對免疫調控的作用建立了細胞工具。 重組質粒經酶切,顯微注射和PCR篩選分別得到了PD-L1的2只Found小鼠和PD-L2的1只Found小鼠,但只有PD-L2Found小鼠可通過生殖器官穩(wěn)定遺傳了PD-L2基因。并且通過southern-blot得到驗證。 本研究通過克隆PD-L1和PD-L2基因來構建穩(wěn)定表達細胞系和轉基因小鼠是成功的。將為進一步研究PD-L1和PD-L2對免疫調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.
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R392.1
本文編號：2483472
[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.
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R392.1
【參考文獻】
相關期刊論文 前1條
1 徐寬楓;B7家族新成員PD-L1、PD-L2的研究進展[J];細胞與分子免疫學雜志;2003年05期
，本文編號：2483472
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