长牡蛎免疫球蛋白超家族（IgSF）成员结构与功能的研究

发布时间：2018-06-17 08:29

  本文选题：长牡蛎 + 免疫球蛋白超家族　； 参考：《中国科学院研究生院(海洋研究所)》2016年博士论文

【摘要】：免疫球蛋白超家族(IgSF)是所有含有免球蛋白样三维拓扑结构的蛋白分子的群体集合。脊椎动物中含有基于免疫球蛋白的适应性免疫,无脊椎动物虽然缺乏高度可变的免疫球蛋白,但已有研究表明无脊椎动物中具有类似适应性免疫的“选择适应性免疫”(alternative adaptive immunity),无脊椎动物中的免疫球蛋白超家族(IgSF)成员分子可能在无脊椎动物的“选择适应性免疫”应答中发挥重要作用。目前对于软体动物IgSF成员结构和免疫功能的了解非常有限。本文采用生物信息学、分子生物学、免疫学等手段,鉴定并分析了长牡蛎Ig SF分子的结构和进化特征,探讨了IgSF分子在长牡蛎个体发育和免疫微生物(河口弧菌、溶藻弧菌、塔氏弧菌、灿烂弧菌和鳗弧菌)和PAMPs(LPS、PGN、GLU和poly(I:C))刺激的表达模式,查明了几种IgSF成员分子(CgJAM-A-L、CgSiglec-1和CgNCAM)的结构与免疫学功能。通过结构域分析,从长牡蛎中共鉴定得到了268个IgSF成员分子,分别含有1-36个Ig结构域,其中162个基因由Ig结构域串联重复而成,而其余106个基因(39.6%)则由Ig结构域与其它结构域重组而成。注释分析提示长牡蛎IgSF主要具有识别和粘附的功能,其中共有217个IgSF成员分子被预测具有识别和结合的功能。HMM-3D功能聚类的分子结果对GO注释的结果做了进一步的补充,将部分在Blast2GO软件中未能成功注释基因与其他明确注释基因聚类到了一起。根据已报道海洋动物基因组中IgSF的进化分析发现,IgSF家族体现出以海绵动物、棘皮动物和半索动物为节点的阶段性扩张趋势。转录组分析发现PAMPs和弧菌刺激后表达量显著升高的IgSF分子数量分别达到116和166个,提示长牡蛎IgSF可能在免疫应答反应中发挥重要功能。发现大量的IgSF在免疫响应中具有高度的特异性,且含较少Ig结构域的IgSF分子功能多样性更为丰富,表明长牡蛎IgSF在进化扩张过程中存在结构和功能的分化。在灿烂弧菌刺激的转录组分析中筛选获得大量的IgSF单碱基突变和可变剪接,并且有大量突变位点具备对灿烂弧菌二次刺激的特异性,暗示长牡蛎IgSF可能通过点突变和可变剪接产生多样性,且点突变形成的新个体变异型可能参与“选择适应性免疫”应答。另外通过分析长牡蛎不同时期的转录组数据发现,部分长牡蛎igsf能够在特定发育时期上调表达,可能参与母源免疫和卵裂期、胚胎期、幼虫期和稚贝型的发育调控。非跨膜型的cgjam-a-l是在长牡蛎中发现的一个jam家族分子同源基因,含有三个串联的i-set型ig结构域。它能够识别并结合多种pamps(lps、pgn、man、lta、poly(i:c)和glu)和微生物(灿烂弧菌、鳗弧菌、大肠杆菌、金黄色葡萄球菌、藤黄微球菌、耶罗维亚酵母和毕赤酵母),并在长牡蛎的固有免疫应答中发挥调理作用。同时cgjam-a-l的结构进化信息表明了jam家族分子与抗体分子可能具有一个多功能的“祖先基因”,并在进化中实现了功能的特异性分化,从而分别形成jam和抗体分子。跨膜型的cgsiglec-1基因包含两个ig结构域和两个胞内的受体酪氨酸抑制基序(itim),并在长牡蛎的免疫识别和诱导下游级联反应中发挥连接作用。它在各组织中广泛分布,并且血淋巴细胞中的转录本能够在灿烂弧菌刺激后显著地上调。cgsiglec-1能够结合多种pamps(psias、lps和pgn),并且通过抑制下游信号通路,在血淋巴细胞的凋亡、吞噬和细胞因子的释放中发挥调节作用。跨膜型的cgncam包含五个ig结构域、两个fniii结构域和一个跨膜区,并可能在长牡蛎的固有免疫中发挥模式识别作用。它与一种植物的凝集素具有较高的相似性,并且在微生物刺激后,血淋巴细胞中的mrna能够显著上调。重组的rcgncam蛋白具有对多种pamps(lps、glu和man)和微生物(灿烂弧菌、鳗弧菌、大肠杆菌、藤黄微球菌、耶罗维亚酵母和毕赤酵母)的广谱识别能力。同时cgncam还可能在长牡蛎extracellulartraps的形成中发挥重要的作用。三中不同类型的igsf共同说明长牡蛎igsf的主要功能是识别和粘附,游离型的igsf可能发挥调理分子的作用,而跨膜型igsf能够作为抗原分子的表面受体并调节下游的免疫通路。综上所述,长牡蛎中的igsf数目众多,种类丰富,并能够在免疫应答中发挥重要作用。长牡蛎igsf可能通过点突变和可变剪接产生基因的多样性,且新形成的个体变异型可能参与“选择适应性免疫”应答。igsf成员分子cgjam-a-l、cgsiglec-1和cgncam能识别结合多种外源微生物,并分别发挥调理分子、免疫调节分子和表面受体的作用。该结果探索了无脊椎动物中igsf分子与高等动物的异质性,初步揭示无脊椎动物IgSF分子在“选择适应性免疫”中的作用,为进一步研究IgSF分子的进化提供了分子生物学证据,丰富和发展了海洋无脊椎动物免疫学内容。
[Abstract]:The immunoglobulin superfamily (IgSF) is a collection of all protein molecules containing a globulin like three-dimensional topology. Vertebrates contain adaptive immunity based on immunoglobulin, and invertebrates, although lack of highly variable immunoglobulin, have shown that invertebrates have similar adaptive immunity. "Alternative adaptive immunity", the immunoglobulin superfamily (IgSF) members of invertebrates may play an important role in the "selective adaptive immune response" response to invertebrates. At present, the understanding of the IgSF member structure and immune function of mollusks is very limited. Informatics, molecular biology and immunology have been used to identify and analyze the structure and evolution characteristics of Ig SF in oysters, and to explore the expression patterns of IgSF molecules in the oysters' ontogeny and immune microorganism (Vibrio estuarine, Vibrio alginolyticus, Vibrio TA, Vibrio resplenden and Vibrio anguinae) and PAMPs (LPS, PGN, GLU and poly (I:C)). Structural and immunological functions of several IgSF members (CgJAM-A-L, CgSiglec-1 and CgNCAM). Through domain analysis, 268 IgSF members were identified from long oysters, containing 1-36 Ig domains, of which 162 genes were repeated in series with the Ig domain, while the rest of the 106 genes (39.6%) were from Ig domain and other junctions. Annotated analysis suggests that oysters IgSF mainly have the function of identification and adhesion, in which a total of 217 IgSF members are predicted to have a functional cluster of functional.HMM-3D, which is identified and combined. The results of the GO annotation are further supplemented by the failure to annotate the genes in the Blast2GO software. According to the reported evolutionary analysis of IgSF in the genome of marine animals, it was reported that the IgSF family showed a phased expansion of the node with spongy, eanthoderma and semi cord animals. The number of IgSF molecules with a significant increase in the expression of PAMPs and Vibrio stimulated by the transcriptional analysis was 11, respectively. 6 and 166, suggesting that the long oyster IgSF may play an important role in the immune response. It is found that a large number of IgSF are highly specific in the immune response, and the functional diversity of the IgSF molecules with less Ig domains is more abundant, indicating the structural and functional differentiation of the long oyster IgSF in the process of evolution and expansion. A large number of IgSF single base and variable splicing were screened in the stimulated transcriptional analysis, and a large number of mutation sites were specific to the two stimulation of Vibrio radivibrio, suggesting that the long oyster IgSF may produce diversity through point mutation and variable splicing, and the new individual variant formed by point mutation may be involved in "selecting adaptive immunity." In addition to the analysis of transcriptional data from different periods of oysters, some long oysters IgSF can be up-regulated at a specific developmental stage and may be involved in developmental regulation of mother source and cleavage, embryo, larval and juvenile type. Non transmembrane cgjam-a-l is a jam family homologous group found in long oysters. It contains three series of i-set type Ig domains. It can identify and combine a variety of PAMPS (LPS, PGN, man, LTA, poly (i:c) and Glu) and microbes (Vibrio, Vibrio eel, Escherichia coli, Staphylococcus aureus, Micrococcus luticus, Pichia Hierro and Pichia pastoris), and play a regulating role in the inherent immune response of long oysters. Cgjam-a-l's structural evolution information indicates that jam family and antibody molecules may have a multi-functional "ancestral gene", and the specific differentiation of functions has been achieved in evolution, thus forming jam and antibody molecules respectively. The transmembrane cgsiglec-1 gene contains two Ig domains and two intracellular receptor tyrosine inhibitors The sequence (ITIM), which plays a connection in the immuno identification and induced cascade reaction of long oysters, is widely distributed in various tissues, and the transcriptional transcript in the blood lymphocytes can be significantly up-regulated after Vibrio splendeni can be combined with a variety of PAMPS (psias, LPS and PGN), and by inhibiting downstream signaling pathways in the blood. Apoptosis, phagocytosis, and cytokine release play an regulatory role. Transmembrane cgncam contains five Ig domains, two FNIII domains and a transmembrane region, and may play a pattern recognition role in the inherent immunity of long oysters. It has a higher similarity with a plant lectin and is stimulated by microbes, MRNA in blood lymphocytes can be significantly up-regulated. The recombinant rcgncam protein has broad spectrum recognition ability for a variety of PAMPS (LPS, Glu and man) and microbes (Vibrio, Vibrio eel, Escherichia coli, Pichia Garcinia, Pichia Hierro and Pichia pastoris). Meanwhile, cgncam may also play an important role in the formation of long oyster extracellulartraps. The main functions of the three different types of IgSF show that the main function of the oyster IgSF is to recognize and adhere, the free type of IgSF may play the role of the modulators, and the transmembrane IgSF can be used as the surface receptor of the antigen molecule and regulate the downstream immune pathway. In summary, the number of IgSF in the oyster is numerous, rich and capable. IgSF may play an important role in the immune response. The variety of gene may be produced by point mutation and alterable splicing, and the newly formed individual variant may participate in the "selective adaptive immune" response to the.Igsf member cgjam-a-l, cgsiglec-1 and cgncam can identify the combination of multiple exogenous microbes and play a regulating molecule, respectively. The effect of immunomodulatory molecules and surface receptors. The results explored the heterogeneity of IgSF and higher animals in invertebrates, preliminarily revealed the role of IgSF in the selection of adaptive immunity, providing molecular biological evidence for further study of the evolution of IgSF molecules and enriching and developing the oceanic spine. The content of animal immunology.
【学位授予单位】：中国科学院研究生院(海洋研究所)
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S917.4
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本文编号：2030396