本文選題：揚(yáng)子鱷 + TCR��； 參考：《中國農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：T細(xì)胞在動(dòng)物適應(yīng)性免疫系統(tǒng)中發(fā)揮重要的作用,主要參與細(xì)胞免疫,具有信號(hào)傳遞、激活B細(xì)胞、殺傷靶細(xì)胞、調(diào)節(jié)免疫應(yīng)答等功能,T細(xì)胞特異性免疫功能的行使需要通過T細(xì)胞表面受體(T cell receptor,TCR)來完成。之前的研究發(fā)現(xiàn)有頌類脊椎動(dòng)物中都存在α、β、γ和δ四種典型的TCR基因類型,其蛋白產(chǎn)物可以形成αβ和γδ兩種跨膜型異源二聚體。此外,在低等哺乳動(dòng)物、鳥類、兩棲類和軟骨魚中還存在非典型的TCR基因類型:TCRμ、VHδ(位于TCRα/δ位點(diǎn)的與免疫球蛋白重鏈可變區(qū)序列相似性更高的V基因)和NAR-TCR。爬行類動(dòng)物在進(jìn)化過程中是連接兩棲類、鳥類和哺乳動(dòng)物的紐帶,目前對(duì)其TCR基因的研究還非常少,在很大程度上限制了我們對(duì)有頜類脊椎動(dòng)物TCR基因進(jìn)化模式和過程的理解。鱷目動(dòng)物是現(xiàn)存較為原始的爬行類動(dòng)物,同時(shí)被廣泛認(rèn)為具有強(qiáng)大的免疫系統(tǒng)。本研究選擇中國特有的揚(yáng)子鱷(Alligator sinensis)為研究對(duì)象,對(duì)其TCR基因進(jìn)行了全面的分析。本研究首先利用鳥類TCR基因恒定區(qū)氨基酸序列在已經(jīng)公布的揚(yáng)子鱷基因組序列中進(jìn)行搜索,得到揚(yáng)子鱷的TCR基因恒定區(qū)的序列信息;再通過篩選揚(yáng)子鱷基因組BAC(bacterial artificial chromosome)文庫,對(duì)得到的含有TCR基因的17條BAC克隆中的9條進(jìn)行全長測序并與基因組序列進(jìn)行拼接,獲得了包含揚(yáng)子鱷TCRβ基因全長(約500Kb)、TCRγ基因全長(109Kb)、TCRα/δ基因大部分V區(qū)和全部C區(qū)(包含兩個(gè)位點(diǎn),位點(diǎn)A跨度約1 Mb,位點(diǎn)B長約400 Kb)的序列信息。結(jié)合RACE(rapid amplification of cDNA ends)實(shí)驗(yàn)獲得的轉(zhuǎn)錄本序列信息和Southern blotting實(shí)驗(yàn)結(jié)果對(duì)獲得的基因組序列進(jìn)行分析并繪制基因位點(diǎn)圖譜,發(fā)現(xiàn)揚(yáng)子鱷TCRβ基因座的結(jié)構(gòu)為 Vβ(39)-Jβ1-ψCβ1-Dβ2-Jβ2(11)-Cβ2-Vβ(4),TCRγ 基因座的結(jié)構(gòu)為 Vγ(18)-Jγ(9)-Cγ。與其他物種相似,揚(yáng)子鱷的TCRδ基因也位于TCRα基因座內(nèi),共有兩個(gè)結(jié)構(gòu)相似的TCRα/δ基因位點(diǎn)。位點(diǎn)A的結(jié)構(gòu)為 V(104)-Dδ1(3)-Jδ1(3)-Cδ1-Jα1(91)-Cα1-VHδ(2)(包含 80 個(gè) Vα/δ 和 26 個(gè) VHδ),位點(diǎn) B 的結(jié)構(gòu)為 V(39)-Dδ2(3)-Jδ2(3)-Cδ2-Vδ-VHδ-Jδ3-ψCδ3-VHδ(3)-Dδ4-Jδ4-Cδ4-VHδ(2)-Dδ5-Jδ5-Cδ5-Jα2(63)-Cα2(包含37個(gè)Vα/δ和11個(gè)VHδ)。RACE實(shí)驗(yàn)證明位點(diǎn)A和位點(diǎn)B是相鄰的,因?yàn)閮蓚�(gè)位點(diǎn)可以共用部分可變區(qū)基因,但基因組序列搜索和BAC克隆篩選暫時(shí)都無法確定其位置關(guān)系。表達(dá)水平的分析發(fā)現(xiàn)揚(yáng)子鱷的所有TCR基因在表達(dá)過程中都不發(fā)生體細(xì)胞超突變(somatic hypermutation,SHM),但其CDR3中存在大量的N/P核苷酸。δ鏈能夠使用至少50%的Vα基因和大部分有功能的VHδ基因,還能同時(shí)使用多個(gè)D基因,使其CDR3的序列和長度多樣性大大增加。定量PCR實(shí)驗(yàn)表明在外周淋巴組織中γδTCR的表達(dá)量與αβ TCR的表達(dá)量基本相同,在小腸上皮等粘膜組織中γδ TCR的表達(dá)量更高。揚(yáng)子鱷的TCRβ位點(diǎn)和TCRγ位點(diǎn)與其他物種在相應(yīng)的基因位點(diǎn)上存在同線性關(guān)系,位點(diǎn)結(jié)構(gòu)和表達(dá)機(jī)制與其他物種基本相同。揚(yáng)子鱷的TCRα/δ位點(diǎn)中存在大量與Cδ共同表達(dá)的VHδ基因,這些VHδ基因主要分為三組:第一組(VHδⅠ)與鳥類中的VHδ相似性較高(VHClanⅠ);第二組(VHδⅡ)與鴨嘴獸和負(fù)鼠中的VHδ和Vμ相似性較高(VH Clan Ⅲ);第三組(VHδⅢ)與鱷目動(dòng)物自身的IGHV(免疫球蛋白重鏈可變區(qū)基因)相似性最高(VH Clan Ⅰ、Ⅱ、Ⅲ中都存在),但多數(shù)為假基因,沒有發(fā)現(xiàn)與Cδ基因共同表達(dá)的轉(zhuǎn)錄本。綜合序列比對(duì)、進(jìn)化分析和同線性分析的結(jié)果,本研究提出了羊膜動(dòng)物TCRα/δ位點(diǎn)的一種可能的進(jìn)化過程:爬行類、鳥類和哺乳類的共同祖先中只存在一個(gè)TCRα/δ位點(diǎn),并且含有VHδ Ⅰ和VHδⅡ基因(VHδ Ⅰ也可能出現(xiàn)在哺乳類與爬行類分化之后),在低等哺乳類中保留了部分VHδ Ⅱ,并以此分化出TCRμ基因,在高等哺乳類中丟失了全部VHδ;爬行類與鳥類的共同祖先中發(fā)生了Cδ基因的復(fù)制(鱷目動(dòng)物的Cδ基因分為三組:CδⅠ、CδⅡ和CδⅢ),鳥類中保留了部分VHδⅠ和CδⅠ,并復(fù)制出一個(gè)新的VHδ-Cδ位點(diǎn);鱷目動(dòng)物保留了 VHδ Ⅰ和VHδ Ⅱ,還出現(xiàn)了 VHδⅢ,并且發(fā)生了一次TCRα/δ位點(diǎn)的整體復(fù)制。揚(yáng)子鱷的TCRα/δ位點(diǎn)B更加保守和古老,與其他物種的TCRα/δ位點(diǎn)存在同線性關(guān)系,位點(diǎn)A是鱷目動(dòng)物出現(xiàn)后由位點(diǎn)B復(fù)制形成的,只保留了 CδⅠ,但TCRα/δ位點(diǎn)A的基因在淋巴組織中的表達(dá)量遠(yuǎn)高于位點(diǎn)B的基因,且位點(diǎn)A的V、J基因數(shù)量更多,在表達(dá)水平上的使用頻率也更高。綜上所述,本論文對(duì)揚(yáng)子鱷TCR基因的位點(diǎn)結(jié)構(gòu)、表達(dá)機(jī)制和進(jìn)化特點(diǎn)等進(jìn)行了多方面的研究,并總結(jié)了羊膜動(dòng)物TCRα/δ位點(diǎn)的進(jìn)化模式,這些研究結(jié)果為探索VHδ基因的起源和TCR基因的進(jìn)化過程提供了重要的線索。
[Abstract]:T cells play an important role in the adaptive immune system of animals. They are mainly involved in cellular immunity, signaling, activating B cells, killing target cells, regulating immune response and so on. The specific immune function of T cells is performed through the T cell surface receptor (T cell receptor, TCR). There are four typical TCR genotypes of alpha, beta, gamma and delta in animals, and their protein products can form two transmembrane heterogenous two polymers of alpha and gamma. In addition, there are atypical TCR gene types in lower mammals, birds, amphibians, and cartilaginous fish: TCR mu, VH Delta (at the TCR alpha / delta loci and the variable sequence of immunoglobulin heavy chains) The higher V gene and the NAR-TCR. reptiles are linked to amphibians, birds and mammals in the evolution process, and the study of their TCR genes is still very few, to a great extent, it restricts our understanding of the evolution patterns and processes of the TCR gene in the vertebrate vertebrates. Reptiles are widely considered to have strong immune systems. This study selected the Chinese alligator (Alligator sinensis) as the research object and analyzed its TCR gene in a comprehensive way. First, the study was conducted by using the sequence of the constant region amino acid of the bird TCR gene in the published genome sequence of the alligator. The sequence information of the TCR gene constant region of the alligator was obtained. By screening the BAC (bacterial artificial chromosome) library of the alligator genome, 9 of the 17 BAC clones containing the TCR gene were fully sequenced and spliced with the genome sequence to obtain the full length (about 500Kb), TCR gamma, containing the TCR beta gene of the alligator Alligator. The whole length (109Kb), the TCR alpha / delta gene most V and all C regions (including two loci, the site A span of about 1 Mb, the site B length about 400 Kb), and the transcriptional sequence information obtained from the RACE (rapid amplification of) experiment and the results of the genomic sequence were analyzed and plotted. The structure of the TCR beta loci of the Chinese alligator was found to be V beta (39) -J beta 1-, C beta 1-D beta 2-J beta 2 (11) -C beta 2-V beta (4), TCR gamma loci were V gamma (18) -J gamma (9). Adadjacent adadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadbecause of the position Some variable region genes can be shared, but genomic sequence search and BAC clone screening can not determine its position relationship temporarily. Analysis of expression level shows that all TCR genes in alligator Alligator do not have somatic hyper mutation (somatic hypermutation, SHM) during the expression process, but there are a large number of N/P nucleotides in CDR3. Delta chains can be found in CDR3. The use of at least 50% of the V alpha gene and most of the functional VH delta genes can also use multiple D genes at the same time, making the sequence and length diversity of the CDR3 greatly increased. Quantitative PCR experiments show that the expression of delta TCR in peripheral lymphoid tissues is basically the same as the expression of alpha beta TCR, and the expression of delta TCR in small intestinal epithelium and other mucosa tissues. The TCR and TCR gamma loci of the alligator and other species have the same linear relationship with other species, and the structure and expression mechanism of the loci are basically the same as those of other species. There are a large number of VH delta genes in the TCR alpha / delta loci of alligator Alligator, which are mainly divided into three groups: the first group (VH delta I) and the birds. The VH delta similarity is higher (VHClan I); the second group (VH Delta II) is similar to the VH Delta and V in the duckbill and the opossum (VH Clan III), and the third group (VH delta III) is the highest (VH Clan, II, and III) of the crocodile itself (VH Clan, II, and III), but most of them are pseudogenes and are not found with the C delta base. A possible evolutionary process of the TCR alpha / delta loci of amniotic amniotic animals was proposed by the co expression of transcriptional transcripts, integrated sequence alignment, evolutionary analysis and linear analysis. The common ancestor of reptiles, birds and mammals had only one TCR alpha / delta locus, and the VH delta I and VH Delta II genes (VH delta I may also appear. After the differentiation of mammals and reptiles), some VH Delta II was retained in the lower mammals, and the TCR gene was differentiated and all VH delta was lost in the higher mammals; the C delta gene was replicated in the common ancestor of reptiles and birds (the C delta gene of the crocodile was divided into three groups: C delta I, C Delta II and C delta), which were retained in birds. Part VH delta I and C delta I, and replicate a new VH Delta -C delta loci; the crocodile retained the VH delta I and VH Delta II, and also appeared VH delta III, and a TCR A / delta locus was produced as a whole. The TCR alpha / delta loci of the alligator are more conservative and old, and have the same linear relationship with the TCR alpha / delta loci of other species, and the loci A is a crocodile movement. Only C delta I was retained after the occurrence of site B, but the expression of TCR alpha / delta locus A in lymphoid tissues is much higher than that of the loci B gene, and the V, J gene number of A and the frequency of use at the expression level are higher. In this paper, the structure, expression mechanism and evolution of the TCR gene in alligator Alligator are described in this paper. Many studies have been carried out and the evolution patterns of TCR alpha / delta loci of amniotic animals have been summarized. These results provide important clues for exploring the origin of VH delta gene and the evolution of TCR genes.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q953
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本文編號(hào)：2056617