【作者】 杨志；

【导师】 赵要风；

【作者基本信息】 中国农业大学 ， 生物化学与分子生物学， 2015， 博士

【摘要】 T细胞在脊椎动物免疫系统中起到非常重要的作用,不仅能够通过特异性的识别结合抗原呈递细胞表面的MHC-抗原复合物来激活B细胞增殖分化并分泌针对该抗原的高亲和性抗体,还可以直接通过活化的细胞毒性T细胞结合并清除抗原物质。T细胞功能的行使需要通过T细胞受体(T cell receptor, TCR)完成,在目前所有的有颌类脊椎动物中都发现了α、β、γ和δ四种典型的TCR基因。鸭是重要的农业经济禽类,近年来在抗病机制方面越来越多地被作为模式动物进行研究,因此对TCR基因结构和表达特点的研究能够为鸭免疫方面的研究提供基础数据和分子生物学依据。首先我们对含有TCR基因的BAC克隆进行筛选,绘制了包含部分V(variable,可变区)基因的基因位点图谱,分析和验证基因座位上的基因。然后利用5’RACE获得大量不同TCR基因的转录本,观察各基因的重组特点。最后通过定量PCR的方法研究不同TCR基因的组织表达特点。所获得BAC克隆上的北京鸭TRB基因座上顺序分布10个V基因(3个为假基因)、1个D(diversity,多样区)基因、2个J-C基因簇及其下游1个转录方向相反的V共计6个J (joining,连接区)基因和2个C (constant,恒定区)基因。11个V基因分为4个基因家族,5’RACE得到的3个BAC上没有的V基因也都属于其中的同一个家族。所有的D、J和C基因都是有功能的,其中2个C基因的胞外结构域高度相似。北京鸭TRG基因位点以典型的易位子形式分布,包括13个V基因(5个为假基因),5个J基因和1个C基因。13个V基因分为6个家族,5’RACE扩增又发现了2个BAC上没有的V基因,也都属于其中的同一个家族。5个J基因都是有功能,并且J4和J5的编码区序列相同。北京鸭的TRA和TRD基因位于同一基因座内,BAC上共分析到了9个Vα基因(3个为假基因)和24个Vδ基因(1个为假基因),分为3个和5个基因家族。5’RACE共发现了BAC上没有的40个Vα基因和6个Vδ基因,都属于BAC上已经发现了的基因家族。在TRA/D基因位点内至少存在68个J。以及各2个Dδ和Jδ基因,从结构上看都是有功能的。北京鸭的第二个TRD基因位点与鸡具有同线性关系,位点内各包括1个V、D、J和C基因。所有TCR基因在V(D)J重组过程中都有明显的P、N核苷酸插入以及重组基因片段末端碱基缺失的现象。统计发现所有5种TCR基因的CDR3长度与之前发现的物种有相同的趋势,但α、β和γ链的CDR3长度变化范围更广。δ链的CDR3长度最长,但稍短于人和小鼠。TRB基因中TRBV3家族的使用频率最高,TRBV1家族没有检测到表达。TRG基因中TRGV1家族的表达量最高,TRGV5家族没有检测到表达。TRA基因有超过一半的转录本使用了TRAV3家族的基因。TRD基因有超过60%的克隆使用了20个Vα基因。此外,我们还发现TRD的等位基因能够大量表达,同时发现TRD基因能够使用0-2个数量不等的D。基因以增加CDR3的多样性。北京鸭的第二个TRD基因在粘膜组织中具有很高的表达水平,可能在粘膜免疫中发挥重要作用。我们在鸡和鸭中都发现存在一种胚系V、J重组,可变区序列类似于TCR和IgL(immunoglobulin light chain, IgL)的基因。该基因在鸡中为单拷贝,在鸭中以9个拷贝串联排列的形式分布。该基因在表达过程中序列不发生突变,组织表达兼具TCR和BCR的特点。鸡的B细胞(DT40)和T细胞(MSB1)都能表达这种TCR-like的基因,免疫荧光的结果将该基因产物定位在MSB1细胞的细胞膜上。综上所述,我们对北京鸭所有5种TCR基因的基因座位、结构特征以及表达特点进行了细致全面的研究,为今后鸭乃至禽类免疫方面的研究提供了重要的基础数据。更多还原

【Abstract】 T cells, which play a major role in regulation and effector functions of immune response, have T-cell receptor (TCR) heterodimers expressed on their surface to react with antigens presented by major histocompatibility complex molecules. So far four typical TCR genes (α,β,γandδ) have been found in jawed vertebrates. As an important poultry, ducks are more and more frequently studied as an avian model for their resistance to various pathogens. So, a thorough analysis of the genomic organization and repertoire diversity of the duck TCR genes may provide basic data and theoretical basis for further understanding of the avian adaptive immune system. In this study, the TRA/D, TRB, and TRG gene loci of Peking duck were mapped by screening a BAC library, and the expressed repertoires of each TCR gene were obtained by5’RACE assay. Finally, the mRNA levels of each TCR gene in different tissues were measured by quantitative PCR.The duck TRB locus in the obtained BAC contains10Vp segments (seven functional and three pseudo genes), a single Dp segment and two functional Cβ genes, which have highly conserved Ig domains and each preceded by two and four functional Jp segments, respectively. Three extra Vβ segments were cloned by5’ RACE assay, and the13Vp gene segments can be grouped into four subgroups. The duck TRG locus has a translocon organization, including13Vγ segments (eight functional and five pseudo genes), five functional Jγ segments, and a single Cγ segment in BAC sequence. Two extra Vγ gene segments were cloned from5’ RACE assay and all15duck Vγ gene segments can be divided into six subgroups. The duck TRD locus is located within the TRA locus, resembling the genomic organization of TRA/D locus in other tetrapods. Within this locus, we identified a single Cα and Cδ gene and33V gene segments, including nine Vα at the5’ end and24Vδ located downstream of the Va group. Three Va and one Vδ segments are pseudogenes. Using5’RACE PCR,40extra Va and six extra Vδ segments were cloned from TRA and TRD cDNA clones. All49(9+40) Va and30(24+6) Vδ segments can be grouped into three and five subgroups, respectively. There are at least68functional Jα gene segments located between the Cδ and Cα genes, and furthermore, two D8and two functional Jδ gene segments were identified upstream of the single Cδ gene, In duck genome sequence, we also identified a second TRD locus, which contains a single VH-like Vδ, Dδ, Jδand Cδ segment. Genes flanking this locus are also conserved in chickens.N and P nucleotide additions and trimming of V, D, and J genes were frequently observed during the V(D)J rearrangement for all four TCR genes. Length distributions of CDR3for all four TCR genes are similar to those of human and mice. The CDR3lengths of TRD chains are longer but constrained, whereas those of the TRA, TRB, and TRG chains are short but variable. For TRB and TRG, the most frequently used V gene subgroup is VpIII and VγI, respectively, whereas the subgroup VβI and VγV, was not observed in all cDNA clones. For TRA and TRD, members of the subgroup VαⅢ occupied more than half of the expressed Va repertoire, and more than60%Vδ repertoire utilized20Va segments. One, two, and none Dδ segments were incorporated within a single CDR3, and a new allele of Dδ2segment was identified in most of the CDR3sequences. We also found that the second TRD gene was highly expressed in mucosal tissue, indicating its important role in mucosal immunity.Based on the genome data of both duck and chicken, we discovered a novel gene of immunoglobulin superfamily characterized by a prejoined VJ in germline sequence. This gene is organized as nine tandem clusters in duck but only a single copy in chicken. V region of this gene shared high sequence identity to V segments of both TCRs and IgLs, and shows no mutations when expressed. This TCR-like gene is highly expressed in both thymus and spleen, as well as in chicken B and T cells lines (DT40and MSB1). Furthermore, the protein encoded by this gene was detected on the surface of MSB1cell by immunofluorescent.In summary, we had thoroughly analysed the genomic organization, the repertoire diversity and V-D-J recombinatorial pattern of five duck TCR genes, these basic data may provide new clues to further understanding of the avian adaptive immune system.更多还原