本文選題：北京鴨 + 免疫球蛋白輕鏈�。� 參考：《中國(guó)農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：鴨是水禽類的代表,具有重要的農(nóng)業(yè)經(jīng)濟(jì)價(jià)值和特殊的免疫學(xué)特點(diǎn),近年來(lái)受到越來(lái)越多免疫學(xué)研究者的關(guān)注。免疫球蛋白(immunoglobulin, Ig)是動(dòng)物適應(yīng)性免疫系統(tǒng)中的關(guān)鍵分子,目前在幾乎所有研究過(guò)的鳥(niǎo)類免疫球蛋白輕鏈(immunoglobulin light chain, IgL)基因位點(diǎn)中,都只有一個(gè)功能Vλ基因片段可以參與VJ重組,因此VJ重組引入的序列多樣性非常有限。鳥(niǎo)類IgL基因主要依賴基因轉(zhuǎn)換(gene conversion)機(jī)制而產(chǎn)生序列多樣性,即利用可變區(qū)假基因作為序列供體來(lái)修飾發(fā)生VJ重組的Vλ基因。兔子及一些偶蹄類動(dòng)物(豬、牛和羊等)的免疫球蛋白基因也能通過(guò)基因轉(zhuǎn)換產(chǎn)生多樣性,但是人和小鼠免疫球蛋白基因幾乎不能發(fā)生基因轉(zhuǎn)換。本研究深入解析了北京鴨IgL基因序列,構(gòu)建了完整的基因位點(diǎn)胚系結(jié)構(gòu)圖譜：系統(tǒng)分析了北京鴨IgL基因多樣性產(chǎn)生機(jī)制的特征,如VJ重組、基因轉(zhuǎn)換、體細(xì)胞超突變等。期望通過(guò)以上研究,使我們對(duì)鴨IgL基因的位點(diǎn)結(jié)構(gòu)和多樣性產(chǎn)生機(jī)制有更全面深入的認(rèn)識(shí)。我們通過(guò)篩選北京鴨基因組細(xì)菌人工染色體(bacterial artificial chromosome, BAC)文庫(kù),并對(duì)篩選到的覆蓋北京鴨完整IgL基因位點(diǎn)的BAC克隆進(jìn)行全長(zhǎng)測(cè)序,獲得了包含北京鴨輕鏈基因的約210 kb的基因組序列。通過(guò)對(duì)該序列的分析,結(jié)合Southern blotting及RACE實(shí)驗(yàn)結(jié)果,最終構(gòu)建了北京鴨IgL基因位點(diǎn)完整胚系結(jié)構(gòu)圖。在北京鴨IgL基因位點(diǎn)中,Cx和Jλ基因?yàn)閱慰截?這與雞等其它鳥(niǎo)類一致；而與其它鳥(niǎo)類不同的是,北京鴨IgL基因位點(diǎn)中共有88個(gè)Vλ基因片段,其中9個(gè)是功能Vλ基因,79個(gè)為假基因。88個(gè)Vλ基因可以分為兩個(gè)家族,而9個(gè)功能Vλ基因同源性較高,屬于同一家族成員。進(jìn)化樹(shù)分析顯示,這9個(gè)功能Vλ基因與其它鳥(niǎo)類的功能Vλ基岡同源性很高,說(shuō)明鴨多個(gè)功能Vλ基因很可能是進(jìn)化過(guò)程中通過(guò)基因復(fù)制產(chǎn)生的。我們通過(guò)特異PCR及序列分析發(fā)現(xiàn),鴨9個(gè)功能Vλ基因均能參與VJ重組過(guò)程。相比于雞等其它鳥(niǎo)類,多個(gè)V基因參與VJ重組明顯增加了鴨IgL基因重組多樣性,但與人和小鼠相比仍然很低。北京鴨VλJλ重組片段中還存在少量N核苷酸和P核苷酸插入。比較北京鴨、人、小鼠和雞的CDRL3區(qū)域的長(zhǎng)度和氨基酸使用情況發(fā)現(xiàn),北京鴨CDRL3長(zhǎng)序列較多且半胱氨酸含量相對(duì)高一些。我們還發(fā)現(xiàn),基因轉(zhuǎn)換在北京鴨IgL基因多樣性中起到重要作用(21日齡北京鴨中,Vλ1、Vx9和Vλ19基因轉(zhuǎn)換頻率為25.3%,平均替換片段長(zhǎng)度為45±41 bp；140日齡北京鴨中為36.5%,平均替換片段長(zhǎng)度為86±46 bp),而且北京鴨9個(gè)功能Vλ基因中都能發(fā)生基因轉(zhuǎn)換,從而可顯著提高北京鴨IgL基因多樣性。為進(jìn)一步探究為什么小鼠不能利用基因轉(zhuǎn)換機(jī)制米增加免疫球蛋白基因的多樣性,我們將上述篩選到的攜帶有完整北京鴨IgL基因位點(diǎn)的BAC克隆進(jìn)行修飾并制備了轉(zhuǎn)基因小鼠。陽(yáng)性轉(zhuǎn)基因小鼠中,外源鴨功能Vλ基因都能參與VJ重組。RNA水平檢測(cè)顯示,各家系小鼠均能正常表達(dá)內(nèi)源小鼠Igλ、Igκ及外源鴨IgL基因。進(jìn)一步的Western blotting實(shí)驗(yàn)表明,轉(zhuǎn)基因小鼠中外源鴨IgL基因在蛋白水平也可以表達(dá)。通過(guò)基因轉(zhuǎn)換分析發(fā)現(xiàn),外源鴨Vλ6、Vλ9和Vx19基因轉(zhuǎn)換頻率僅為2.69%,雖然高于野生型小鼠(小鼠IgG,0.1%),但低于北京鴨自身中Vλ6、Vλ9和Vλ19基因轉(zhuǎn)換頻率。由此我們推測(cè),野生型小鼠免疫球蛋白基因不發(fā)生基因轉(zhuǎn)換,可能是受其基因結(jié)構(gòu)和體內(nèi)分子微環(huán)境共同影響而造成的。綜上所述,我們首次獲得了鴨IgL基因位點(diǎn)完整胚系結(jié)構(gòu)圖,并對(duì)其多樣性產(chǎn)生機(jī)制如VJ重組、體細(xì)胞超突變、基因轉(zhuǎn)換等進(jìn)行了詳細(xì)研究分析,為人們理解鴨免疫球蛋白基因的多樣性產(chǎn)生機(jī)制奠定了基礎(chǔ)。
[Abstract]:As a representative of waterfowl, duck has important agricultural economic value and special immunological characteristics. In recent years, more and more immunology researchers have paid more attention to it. Immunoglobulin (Ig) is the key element in the adaptive immune system of animals. At present, the immunoglobulin light chain (immunoglobuli In the n light chain, IgL) gene loci, only one functional V lambda gene fragment can participate in VJ recombination, so the sequence diversity of VJ recombination is very limited. The avian IgL gene mainly relies on the gene conversion (gene conversion) mechanism to produce sequence diversity, which means that the variable region pseudogenes are used as the sequence donor to modify the VJ recombination. The V lambda gene. The immunoglobulin genes of rabbits and some even hoofed animals (pigs, cattle and sheep, etc.) can also produce diversity through gene conversion, but human and mouse immunoglobulin genes can hardly have genetic transformation. This study has deeply analyzed the IgL gene sequence of Beijing duck, and constructed a complete structure map of the germ line of the gene loci. The characteristics of the genetic diversity of the Beijing duck IgL gene, such as VJ recombination, gene transformation, and somatic hyper mutation, are analyzed. We expect to make a more thorough understanding of the mechanism of the site structure and diversity of the duck IgL gene through the above study. We have screened the artificial chromosome of the genome of the duck genome of Beijing duck (bacterial artific). Ial chromosome, BAC) library and full-length sequencing of the screened BAC clones covering the complete IgL gene loci of Beijing duck, and obtained the genome sequence of about 210 KB containing the Beijing duck light chain gene. Through the analysis of this sequence and combining the results of Southern blotting and RACE experiment, the whole embryo of the IgL gene locus of Beijing duck was finally constructed. In the IgL gene site of the Beijing duck, the Cx and J lambda based loci are consistent with other birds such as chicken. Unlike other birds, there are 88 V lambda genes in the Beijing duck IgL gene locus, of which 9 are functional V lambda, and 79 pseudogenes.88 V lambda genes can be divided into two families and 9 function V lambda genes. High homology belongs to the same family. Evolutionary tree analysis shows that these 9 functional V lambda genes are highly homologous to the functional V lambda Keegan of other birds, indicating that the multiple functional V lambda gene of ducks is likely to be produced by gene replication during evolution. We found that the 9 functional V lambda genes of ducks can all participate in VJ through specific PCR and sequence analysis. Compared with other birds such as chickens, multiple V genes involved in VJ recombination significantly increased the recombinant diversity of duck IgL gene, but still very low compared to human and mouse. A small amount of N nucleotides and P nucleotides were inserted in the recombinant fragment of Beijing duck V lambda J lambda. Comparison of the length and amino acid use of the CDRL3 region of Beijing duck, human, rat and chicken. It was found that the CDRL3 long sequence of the Beijing duck was more and the cysteine content was relatively high. We also found that gene conversion plays an important role in the IgL gene diversity of Beijing duck (21 day old Beijing ducks, the conversion frequency of V lambda 1, Vx9 and V lambda 19 is 25.3%, the average replacement fragment length is 45 + 41 BP, and 140 days old Beijing duck is 36.5%, with an average replacement. " The length of the fragment was 86 + 46 BP), and the gene conversion in the 9 functional V lambda gene of Beijing duck could be produced, which could significantly improve the diversity of the IgL gene in Beijing duck. To further explore why the mice could not use the gene conversion mechanism to increase the diversity of the immunoglobulin gene, we carried the above screening of the complete Beijing duck Ig The BAC clone of the L gene site was modified and the transgenic mice were prepared. In the positive transgenic mice, the exogenous duck function V lambda gene could all participate in the VJ recombinant.RNA level detection. All the mice were able to express the endogenous mouse Ig lambda, Ig kappa and the exogenous duck IgL gene. The further Western blotting experiment showed that the transgenic mice were exogenous. The duck IgL gene could also be expressed at the protein level. Through gene transformation analysis, the conversion frequency of V lambda 6, V lambda 9 and Vx19 gene was only 2.69%, although it was higher than that of wild type mice (mice IgG, 0.1%), but lower than that of V lambda 6, V lambda 9 and V lambda 19 gene in Beijing duck itself. Genetic transformation may be caused by the common influence of its gene structure and molecular microenvironment. In summary, we have first obtained the structure map of the complete embryo line of the duck IgL gene site, and detailed analysis of its diversity mechanism such as VJ recombination, somatic hyper mutation, gene conversion and so on. The mechanism of the diversity of protein genes has laid a foundation.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S834.81
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本文編號(hào)：2043483