【摘要】：PD-L1、PD-L2是新發(fā)現(xiàn)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(guò)與受體PD-1結(jié)合抑制T細(xì)胞的增殖和過(guò)度活化,在細(xì)胞免疫應(yīng)答過(guò)程中起負(fù)調(diào)控作用,同時(shí)通過(guò)影響細(xì)胞因子的分泌對(duì)體液免疫也發(fā)揮了調(diào)控作用。PD-L1、PD-L2/PD-1路徑在機(jī)體自身免疫耐受和腫瘤細(xì)胞逃逸機(jī)體免疫監(jiān)控過(guò)程中都發(fā)揮著重要作用。 本研究通過(guò)RT-PCR的方法成功地從PHA刺激的小鼠外周血單個(gè)核細(xì)胞和小鼠胸腺組織中克隆了PD-L1和PD-L2基因,并構(gòu)建了重組真核表達(dá)載體pEF6/PD-L1-V5 His和pEF6/PD-L2-V5 His。兩個(gè)重組質(zhì)粒轉(zhuǎn)染CHO細(xì)胞,經(jīng)間接免疫熒光實(shí)驗(yàn)證明了構(gòu)建的重組質(zhì)粒可以CHO細(xì)胞中的成功表達(dá)PD-L1和PD-L2基因。穩(wěn)定篩選后,經(jīng)western-blot檢測(cè)得到了穩(wěn)定表達(dá)PD-L1和PD-L2的細(xì)胞株,為體外研究PD-L1和PD-L2對(duì)免疫調(diào)控的作用建立了細(xì)胞工具。 重組質(zhì)粒經(jīng)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(guò)生殖器官穩(wěn)定遺傳了PD-L2基因。并且通過(guò)southern-blot得到驗(yàn)證。 本研究通過(guò)克隆PD-L1和PD-L2基因來(lái)構(gòu)建穩(wěn)定表達(dá)細(xì)胞系和轉(zhuǎn)基因小鼠是成功的。將為進(jìn)一步研究PD-L1和PD-L2對(duì)免疫調(diào)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(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392.1

【參考文獻(xiàn)】

相關(guān)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(jìn)展[J];細(xì)胞與分子免疫學(xué)雜志;2003年05期

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