【作者】 岳挺；

【导师】 李厚达；

【作者基本信息】 扬州大学 ， 动物学， 2005， 硕士

【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。更多还原

【Abstract】 PD-Ll and PD-L2 are two co-stimulate molecule ligands found recent years .which belong to B7 family. They share the same receptor PD-1 .By the interaction with PD-1 they affect activated T cells and diminish their proliferation and cytokine production. The engagement of two ligands to the receptor PD-1 result in a negative regulatory effect in T cell response. On the other hand they can also regulate B cell response though the effect on cytokine production. The PD-Ll and PD-L2/PD-1 pathway is not only take an important role in peripheral self tolerance but also found act on the process of tumor cells evading immune systems.In this research we cloned encoding sequence of PD-L1 and PD-L2 by RT-PCR from the total RNA isolated from PHA stimulated mouse peripheral blood mononuclear cells and the total RNA isolated from mouse thymus. After sequencing we found that the sequence of cloned PD-Ll is exactly the same as Genebanks while the sequence of cloned PD-L2 has one mutation. We consult reference and consider it not important.We successfully construct two recombinant eukaryotic expression vectors pEF6/-PD-Ll-V5 His and pEF6/-PD-Ll-V5 His which contain the target genes. Then the recombinant vectors were transfected into CHO cells separately for transient expression and the expression was identified by immunofluorescence technology. We also obtained the stable expression cell lines through the selection of the transfected cells and was identified using western-blot. These suggest that these cell lines, which stably express PD-Ll or PD-L2, can be strongly useful tools to study the immune response regulated by PD-L2 in vitro.We got the DNA fragments for microinjection by restriction endonuclease digestion. Using microinjection into male nuclei two founder transgenic mice of PD-L1 and one of PD-L2 was established after PCR detection. But only PD-L2 founder mouse can passage the transgene to its offspring and was identified by southern-blot.All these results suggest that the construction of stable cell lines and transgenic mouse are successful and will be strongly useful tools to study the function of PD-Ll and PD-L2 in vitro and vivo.更多还原