北京鸭免疫球蛋白轻链基因胚系结构及多样性产生机制研究

发布时间：2018-06-20 07:28

  本文选题：北京鸭 + 免疫球蛋白轻链　； 参考：《中国农业大学》2016年博士论文

【摘要】：鸭是水禽类的代表,具有重要的农业经济价值和特殊的免疫学特点,近年来受到越来越多免疫学研究者的关注。免疫球蛋白(immunoglobulin, Ig)是动物适应性免疫系统中的关键分子,目前在几乎所有研究过的鸟类免疫球蛋白轻链(immunoglobulin light chain, IgL)基因位点中,都只有一个功能Vλ基因片段可以参与VJ重组,因此VJ重组引入的序列多样性非常有限。鸟类IgL基因主要依赖基因转换(gene conversion)机制而产生序列多样性,即利用可变区假基因作为序列供体来修饰发生VJ重组的Vλ基因。兔子及一些偶蹄类动物(猪、牛和羊等)的免疫球蛋白基因也能通过基因转换产生多样性,但是人和小鼠免疫球蛋白基因几乎不能发生基因转换。本研究深入解析了北京鸭IgL基因序列,构建了完整的基因位点胚系结构图谱：系统分析了北京鸭IgL基因多样性产生机制的特征,如VJ重组、基因转换、体细胞超突变等。期望通过以上研究,使我们对鸭IgL基因的位点结构和多样性产生机制有更全面深入的认识。我们通过筛选北京鸭基因组细菌人工染色体(bacterial artificial chromosome, BAC)文库,并对筛选到的覆盖北京鸭完整IgL基因位点的BAC克隆进行全长测序,获得了包含北京鸭轻链基因的约210 kb的基因组序列。通过对该序列的分析,结合Southern blotting及RACE实验结果,最终构建了北京鸭IgL基因位点完整胚系结构图。在北京鸭IgL基因位点中,Cx和Jλ基因为单拷贝,这与鸡等其它鸟类一致；而与其它鸟类不同的是,北京鸭IgL基因位点中共有88个Vλ基因片段,其中9个是功能Vλ基因,79个为假基因。88个Vλ基因可以分为两个家族,而9个功能Vλ基因同源性较高,属于同一家族成员。进化树分析显示,这9个功能Vλ基因与其它鸟类的功能Vλ基冈同源性很高,说明鸭多个功能Vλ基因很可能是进化过程中通过基因复制产生的。我们通过特异PCR及序列分析发现,鸭9个功能Vλ基因均能参与VJ重组过程。相比于鸡等其它鸟类,多个V基因参与VJ重组明显增加了鸭IgL基因重组多样性,但与人和小鼠相比仍然很低。北京鸭VλJλ重组片段中还存在少量N核苷酸和P核苷酸插入。比较北京鸭、人、小鼠和鸡的CDRL3区域的长度和氨基酸使用情况发现,北京鸭CDRL3长序列较多且半胱氨酸含量相对高一些。我们还发现,基因转换在北京鸭IgL基因多样性中起到重要作用(21日龄北京鸭中,Vλ1、Vx9和Vλ19基因转换频率为25.3%,平均替换片段长度为45±41 bp；140日龄北京鸭中为36.5%,平均替换片段长度为86±46 bp),而且北京鸭9个功能Vλ基因中都能发生基因转换,从而可显著提高北京鸭IgL基因多样性。为进一步探究为什么小鼠不能利用基因转换机制米增加免疫球蛋白基因的多样性,我们将上述筛选到的携带有完整北京鸭IgL基因位点的BAC克隆进行修饰并制备了转基因小鼠。阳性转基因小鼠中,外源鸭功能Vλ基因都能参与VJ重组。RNA水平检测显示,各家系小鼠均能正常表达内源小鼠Igλ、Igκ及外源鸭IgL基因。进一步的Western blotting实验表明,转基因小鼠中外源鸭IgL基因在蛋白水平也可以表达。通过基因转换分析发现,外源鸭Vλ6、Vλ9和Vx19基因转换频率仅为2.69%,虽然高于野生型小鼠(小鼠IgG,0.1%),但低于北京鸭自身中Vλ6、Vλ9和Vλ19基因转换频率。由此我们推测,野生型小鼠免疫球蛋白基因不发生基因转换,可能是受其基因结构和体内分子微环境共同影响而造成的。综上所述,我们首次获得了鸭IgL基因位点完整胚系结构图,并对其多样性产生机制如VJ重组、体细胞超突变、基因转换等进行了详细研究分析,为人们理解鸭免疫球蛋白基因的多样性产生机制奠定了基础。
[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.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S834.81
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本文编号：2043483