“PQRSPT”基序在人B7-H3基因生物学功能中的分子机制研究

发布时间：2018-01-11 00:11

本文关键词：“PQRSPT”基序在人B7-H3基因生物学功能中的分子机制研究　出处：《苏州大学》2011年硕士论文　论文类型：学位论文

【摘要】：T细胞的有效活化需要双信号,其中共刺激分子在免疫调节中发挥着重要的作用,B7-H3是共刺激分子B7家族成员之一。人B7-H3有两种不同形式的剪切体:2IgB7-H3和4IgB7-H3。2IgB7-H3胞外段由IgV-IgC两个免疫球蛋白结构域组成,而4IgB7-H3胞外段由IgV1-IgC1-IgV2-IgC2四个免疫球蛋白结构域组成。研究报道发现4IgB7-H3是人各组织细胞中B7-H3的主要表达形式,目前仍缺乏对两种剪切体的差异研究。许多共刺激分子分别以细胞膜型和可溶型两种形式存在。本实验室在健康人外周血和组织液中发现存在天然形式的sB7-H3,且发现sB7-H3仅源于2IgB7-H3。基因序列研究发现4IgB7-H3胞外段4个结构域是由编码2IgB7-H3 IgV和IgC外显子复制的结果,2IgB7-H3和4IgB7-H3两种剪切体的同源性超过95%,本研究通过氨基酸序列比对和分析,发现在人4IgB7-H3分子第一个C样免疫球蛋白结构域碳端末端存在一段独特的氨基酸序列为“PQRSPT”,其在2IgB7-H3分子序列中缺失。本研究以此为出发点获得了增加“PQRSPT”基序的2IgB7-H3-Add基因和缺失“PQRSPT”基序的4IgB7-H3-Del基因,并构建相应的基因转染细胞株,以此来探讨“PQRSPT”基序在人B7-H3基因生物学功能及可溶性B7-H3产生过程中的分子机制。一、2IgB7-H3-Add与4IgB7-H3-Del基因转染细胞株的建立 RT-PCR法分别扩增2IgB7-H3与4IgB7-H3编码区全长基因,并通过拼接PCR的方法获得增加“PQRSPT”基序的2IgB7-H3-Add基因和缺失“PQRSPT”基序的4IgB7-H3-Del基因。将两个目的基因片段双酶切后与pIRES2-EGFP真核表达载体连接,构建重组子pIRES2-EGFP/2IgB7-H3-Add和pIRES2-EGFP/4IgB7-H3- Del,经测序鉴定正确后通过脂质体转染法将两个重组载体分别导入小鼠L929细胞。RT-PCR结果表明表明导入的外源性2IgB7-H3-Add和4IgB7-H3-Del基因已分别成功整合到L929细胞基因组中并能成功地进行转录;通过流式细胞术检测发现GFP和B7-H3蛋白在两株细胞表面均稳定高表达;Western Blot分析结果显示L929/2IgB7-H3-Add和L929/4IgB7-H3-Del细胞均能成功表达B7-H3蛋白。二、“PQRSPT”基序在人B7-H3基因生物学功能中的分子机制研究收集上述基因转染细胞的培养上清进行ELISA和Western Blot分析,结果显示在2IgB7-H3和4IgB7-H3-Del基因转染细胞培养上清中存在可溶性B7-H3蛋白,而4IgB7-H3和2IgB7-H3-Add基因转染细胞培养上清与对照组一样,都未检测出可溶性形式的存在。该研究结果表明保守性氨基酸“PQRSPT”可能是人4IgB7-H3不能形成可溶性形式的一个重要基序。同时,基因转染细胞与外周血T细胞共培养的淋巴细胞增殖实验及分泌细胞因子的检测实验结果发现,2IgB7-H3基因转染细胞能显著地促进T细胞体外增殖与细胞因子IL-2和IFN-γ的分泌,相比之下,增加“PQRSPT”基序的2IgB7-H3-Add基因转染细胞则无此效应;4IgB7-H3基因转染细胞能显著抑制T细胞的增殖与细胞因子IL-2和IFN-γ的分泌,而缺失“PQRSPT”基序的4IgB7-H3-Del基因转染细胞则无明显作用。以上研究结果均证实“PQRSPT”基序可能是4IgB7-H3分子中的功能结构域,且对B7-H3生物学功能的改变具有重要作用。综上所述,本实验成功构建了L929/2IgB7-H3-Add和L929/ 4IgB7-H3-Del两株转基因细胞,对基因转染细胞株培养上清中sB7-H3的检测结果表明了“PQRSPT”基序与人4IgB7-H3分子不能分泌可溶性形式密切相关。T细胞体外增殖实验显示,两株基因转染细胞均对T细胞的增殖和细胞因子的分泌无效应,表明“PQRSPT”基序可能是4IgB7-H3分子发挥抑制作用的重要基序。上述结果为进一步阐明sB7-H3的来源机制提供新的思路,以及为研究人B7-H3两种剪切体的差别性奠定了分子基础。
[Abstract]:Effective activation of the T cell requires two signals. The costimulatory molecules play an important role in immune regulation, B7-H3 is one of the costimulatory molecules of B7 family members. B7-H3 there are two different forms of shear: 2IgB7-H3 and 4IgB7-H3.2IgB7-H3 extracellular domain is composed of IgV-IgC two immunoglobulin domains, 4IgB7-H3 cell the outer segment consists of IgV1-IgC1-IgV2-IgC2 four immunoglobulin domains. Studies suggest that 4IgB7-H3 is the main form of expression of B7-H3 in various tissues, there is still a lack of study on the differences of the two variants.
Many costimulatory molecules exist respectively in cell membrane and soluble forms. There are two natural forms of sB7-H3 were found in our laboratory in healthy human peripheral blood and tissue fluid, and found that sB7-H3 only from the sequence of 2IgB7-H3. gene showed that 4IgB7-H3 extracellular domain 4 is copied by the code 2IgB7-H3 IgV and IgC, 2IgB7-H3 and 4IgB7-H3 two variants homology of more than 95%, the amino acid sequence alignment and analysis, found in human 4IgB7-H3 molecules first C like immunoglobulindomains carbon terminal has a unique amino acid sequence "PQRSPT", and its deletion in 2IgB7-H3 molecules sequence. This study as a starting point for the "PQRSPT" motif of the 2IgB7-H3-Add gene and the deletion of "PQRSPT" motif of 4IgB7-H3-Del gene, and construct the corresponding gene transfected cell line, in order to explore the The "PQRSPT" motif is a molecular mechanism in the biological function of human B7-H3 gene and the production of soluble B7-H3.
The establishment of 2IgB7-H3-Add and 4IgB7-H3-Del gene transfected cell lines
Full length 2IgB7-H3 were amplified with 4IgB7-H3 gene encoding RT-PCR and PCR method, by splicing method to obtain "PQRSPT" motif of the 2IgB7-H3-Add gene and the deletion of "PQRSPT" motif of the 4IgB7-H3-Del gene. The two gene fragments were digested with pIRES2-EGFP eukaryotic expression vector, recombinant pIRES2-EGFP/2IgB7-H3-Add and pIRES2-EGFP/4IgB7-H3- Del, after identification by sequencing by Lipofectamine two recombinant plasmids were respectively transfected into.RT-PCR L929 cells in mice results showed that exogenous 2IgB7-H3-Add and 4IgB7-H3-Del gene were successfully integrated into the genome of L929 cells and successfully through transcription; flow cytometry showed that GFP and B7-H3 protein in two strains the cell surface was stable and high expression; Western Blot analysis showed that L929/2IgB7-H3-Add and L929/4IgB7-H3-Del cells B7-H3 protein can be successfully expressed.
Two, study on the molecular mechanism of "PQRSPT" motif in human B7-H3 gene biological function
Culture supernatants were collected the transfected cells was analyzed by ELISA and Western Blot, showed the presence of soluble B7-H3 protein in the supernatant of cultured in 2IgB7-H3 and 4IgB7-H3-Del transfected cells, while 4IgB7-H3 and 2IgB7-H3-Add gene transfected cell culture supernatant as control group, were not detected in soluble form. The results of the study indicate that conservative amino acid "PQRSPT" may be an important motif of 4IgB7-H3 cannot form a soluble form. At the same time, lymphocytes were cultured transfected cells and peripheral blood T cell proliferation and cytokine secretion in experimental detection experimental results showed that the 2IgB7-H3 gene transfected cells could significantly promote cell proliferation and secretion of T cytokines IL-2 and IFN- gamma in contrast, the increase of "PQRSPT" motif of 2IgB7-H3-Add gene transfected cells had no such effect; 4IgB7-H3 gene transfection Secretory cells can significantly inhibit the proliferation of T cells and cytokines IL-2 and IFN- gamma, but the lack of a "PQRSPT" motif of 4IgB7-H3-Del gene transfected cells has no obvious effect. The above results demonstrated that the "PQRSPT" motif may be the functional domain of 4IgB7-H3 molecules, and the biological function of B7-H3 has great change. In summary, we successfully constructed L929/2IgB7-H3-Add and L929/ 4IgB7-H3-Del two transgenic cells. The detection results of culture supernatant of sB7-H3 transfected cells showed a "PQRSPT" motif and 4IgB7-H3 molecules cannot display the secreted soluble forms of.T is closely related to cell proliferation in vitro, two strains were transfected cells on T cells the proliferation and cytokine secretion had no effect, that the "PQRSPT" motif may be an important motif in 4IgB7-H3 molecules play inhibition. The above results It provides a new idea for further clarifying the source mechanism of sB7-H3 and laying a molecular basis for the study of the difference between the two kinds of human B7-H3 shear bodies.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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