五龙鹅MHC ClassⅠ基因克隆及同源建模研究

主要组织相容性复合体(MHC)与动物机体对外源性抗原的免疫应答之间存在关联。从GenBank/DDBJ/EMBL基因库中读取鸡、其他鸟类、爬行类和哺乳类的MHC ClassⅠ基因进行序列分析设计引物, 使用LA-PCR法从五龙鹅的基因组中克隆了MHC ClassⅠ基因序列(DNA序列和mRNA序列GenBank登录号分别为: AM114925和AM114924), 并分析其基因组结构。运用生物信息学技术对测序结果进行分析显示: 基因组DNA由8个外显子和7个内含子组成, 与鸡基因序列同源率为60.8%~64.1%, 与人的同源率为42.9%。分子进化树进一步揭示了五龙鹅与鸡、其他鸟类、爬行类、哺乳类以及人类的进化关系, 同源建模分析发现该基因由氨基末端结构域和羧基末端结构域构成。     

Class I MHC polymorphism and evolution in endangered California Chinook and other Pacific salmon

Twelve MHC class I exon 2 sequences were uncovered in a sample from the endangered Sacramento River winter-run Chinook salmon in the central valley of California. Phylogenetic analysis of the 12 sequences indicates that the alleles descend from two of six major allelic lineages found among four Pacific salmon species. Nine of the 12 alleles belong to an allelic lineage that began diversifying 8 million years ago, just prior to the estimated time of Chinook speciation. The most recent common ancestor of all 12 winter-run alleles is estimated to be 15 million years ago, approximately 5 million years before the radiation of the Pacific salmon species. The average nonsynonymous distance among the peptide binding-region codons of exon 2 for the 12 alleles is significantly higher than the average synonymous distance in these codons. We estimate the symmetrical overdominant selection coefficient against homozygotes for this exon to be 0.038. Thus, strong positive and balancing selection has maintained functional diversity in the peptide-binding region of the exon over millions of years and this variation has not yet been substantially eliminated by increased genetic drift due to the recent dramatic decline in abundance of this Chinook salmon population.     

Class II MHC: sweetening the peptide only diet?

MHC molecules typically bind peptides to create ligands for the T cell antigen receptor. In this issue of Cell, report an unexpected association of class II MHC molecules with processed zwitterionic polysaccharides from pathogenic bacteria. The complexes appear to modulate the T cell dependent pathology of abscess formation.     

MHC Class Ib grc—G／C基因的研究进展

ＭＨＣ的ｃｌａｓｓ基因,可分为经典的ｃｌａｓｓ Ｉ和非经典的ｃｌａｓｓＩ,后者称ｃｌａｓｓ Ｉｂ。ｃｌｓｓ Ｉ与β／β Ｔ细胞相关在细胞表面高密度表达,ｃｌａｓｓ Ｉｂ在细胞表面弱表达,其功能尚不清楚,ｃｌａｓｓ Ｉｂ的基因区域中含有一个ｇｒｃ区域,ｇｒｃ基因的ｒｃｃ,ｆｔ,ｄｗ－３三组基因组成。     

Exploiting the Peptide — MHC Water Interface in the Computer-Aided Design of Non-Natural Peptides that Bind to the Class I MHC Molecule HLA-A2

Abstract

Class I major histocompatibility complex (MHC) molecules bind peptides derived from intra-cellular proteins and present them to cytotoxic T cells. Certain human immunological diseases are associated with errors in this process. Here we describe an approach to the design of non-natural peptides that could potentially interfere with peptide presentation associated with autoimmune diseases. We have shown previously that the interaction of the peptide GILGFVFTL with the MHC molecule HLA-A2 is mediated by a network of water molecules. In principle, the addition of hydroxyl groups to the peptide could allow for an enhanced interaction of the modified peptide with this water network. Here we illustrate this approach using a peptide having the non-natural amino acid homoserine at position 3, GIhSGFVFTL, and also peptides in which the Cα(F5)—CO—NH1—Cα(V6) peptide bond is replaced by an ether. Cα(F5)—CH(X)—O—Cα(V6), to give the non-natural peptide GILGF—CH(X)—O—VFTL, where X = CH₂OH or CH₃. In a 200 ps solvated molecular dynamics simulation of the HLA-A2 complexes of each peptide for GIhSGFVFTL and GILGF—CH(CH₂OH)—O—VFTL the peptide conformation remained essentially unchanged from that of GILGFVFTL in the X-ray structure of its complex with HLA-A2. In contrast, for GILGF—CH(CH₃)—O—VFTL the peptide conformation deviated from the X-ray conformation, indicating the importance of the hydroxyl group.     

绵羊基因组MHC ClassⅢ区段部分基因的分离与鉴定

绵羊基因组MHC段分为ClassⅠ、Class Ⅲ和ClassⅡ（含Ⅱa和Ⅱb两个亚区）3个区段,与另外2个区段相比,Class Ⅲ区的基因信息远少于ClassⅠ和ClassⅡ区.为丰富绵羊基因组 MHC Class Ⅲ 区段基因信息. 本研究用位于中国美利奴羊基因组BAC文库中 MHC ClassⅢ 区段4个BAC克隆的酶切片段制备32P 标记探针,继而采用噬菌斑原位杂交筛选法筛选中国美利奴羊混合组织cDNA 文库,并对分离到的cDNA 阳性克隆进行全序列测定及生物信息学分析.本实验共筛选出 31 个 cDNA 阳性克隆,对其序列进行了测定及分析,确定了序列在ClassⅢ 区段上的位置,并通过在NCBI中的同源检索对其功能做了初步鉴定.由实验可知,利用BAC 文库与 cDNA 文库杂交筛选法对较大区段基因的筛选和分离是有效的.同时,对分离到的表达序列结合生物信息学进行分析,这将有助于对该序列功能的深入研究.     

三种猫科动物MHC Class Ⅱ DRB等位基因序列变异性分析

分析了云豹(Neofelisnebulosa)、豹(Pantherapardus)和东北虎(Pantheratigrisaltaica)等3种猫科动物的主要组织相容性复合物ClassⅡDRB座位的等位基因序列变异性。使用一对简并性引物扩增了DRB座位第二外元目标片段。用单链构像多态性分析方法确定不同的单倍型。每个个体挑出15个单克隆用来分离、纯化和测序。实验中从4个个体中获得了8种不同序列。237bp核苷酸序列中发现有59个变异位点。根据人类的抗原肽结合区推测79个氨基酸位点中存在21个假定的抗原肽结合位点,而且非同义替换率明显高于同义替换率,这可能说明了第二外元的较高变异性是由平衡选择作用来维持的。重建的NJ树和MP树显示豹和东北虎的亲缘关系较近,而两者与云豹的亲缘关系较远。     

MHC Class I Exon 4 in the Multiocellated Racerunners （Eremias multiocellata）： Polymorphism,Duplication and Selection

The major histocompatibility complex （MHC） is a dynamic genetic region with an essential role in the adaptive immunity of jawed vertebrates. The MHC polymorphism is affected by many processes such as birth-and- death evolution, gene conversion, and concerted evolution. Studies investigating the evolution of MHC class I genes have been biased toward a few particular taxa and model species. However, the investigation of this region in nonavian reptiles is still in its infancy. We present the first characterization of MHC class I genes in a species from the family Lacertidae. We assessed genetic diversity and a role of selection in shaping the diversity of MHC class I exon 4 among 37 individuals of Eremias multiocellata from a population in Lanzhou, China. We generated 67 distinct DNA sequences using cloning and sequencing methods, and identified 36 putative functional variants as well as two putative pseudogene-variants. We found the number of variants within an individual varying between two and seven, indicating that there are at least four MHC class I loci in this species. Gene duplication plays a role in increasing copy numbers of MHC genes and allelic diversity in this species. The class I exon 4 sequences are characteristic of low nucleotide diversity. No signal of recombination is detected, but purifying selection is detected in β2-microglobulin interaction sites and some other silent sites outside of the function-constraint regions. Certain identical alleles are shared by Eremias multiocellata and E. przewalskii and E. brenchleyi, suggesting trans-species polymorphism. The data are compatible with a birth-and-death model of evolution.     

病毒干扰MHCⅠ类抗原呈递策略的研究进展

细胞表面MHCⅠ类分子在CTL的产生和作为CTL受体的配体清除病毒感染细胞中发挥着重要作用。因此,许多病毒在其生活周期的不同阶段干扰MHCⅠ类抗原呈递并不足为奇。深入理解病毒利用的干扰策略不仅有助于揭示病毒的致病机理,而且有助于制定新的对策避免病毒逃逸,这些研究最终可能建立有效控制病毒感染的免疫疗法。因此,本文将就病毒干扰MHCⅠ类抗原呈递策略的研究进展做一综述。     

玉米中Class Ⅰ和Class ⅡNAS蛋白之间的BiFC互作研究

烟草胺合成酶（nicotianamine synthase,NAS）能够催化合成植物体内铁运输所需的螯合物烟草胺（nicotianamine,NA）,在植物维持铁稳态方面发挥重要的作用。玉米、小麦和大麦等禾本科植物的NAS蛋白进化为ClassⅠ和ClassⅡ两个亚家族,可能分别参与调节铁的吸收和运输,其家族成员之间蛋白序列同源性较高,ClassⅡNAS具有特异的N端可变结构域。通过进化分析分析玉米NAS的两个亚家族,以及建模预测两类亚家族代表基因ZmNAS1(ClassⅠ)和ZmNAS3(ClassⅡ)的蛋白结构,结果表明ZmNAS1和ZmNAS3的三维结构高度相似,推测可能通过同源或异源二聚体化发挥功能;进一步通过双分子荧光互补（bimolecular fluorescence complementation,BiFC）分析ZmNAS1和ZmNAS3的相互作用,结果表明,ZmNAS1和ZmNAS3蛋白可以互作,删除N端可变结构域的ZmNAS3?N只能与ZmNAS3蛋白互作而不与ZmNAS1互作,推测ZmNAS可以形成同源二聚体,而形成异源二聚体需要ClassⅡ家族蛋白的N端可变结构域...     

Localized Populations of CD8low/? MHC Class I Tetramer+ SIV-Specific T Cells in Lymphoid Follicles and Genital Epithelium

CD8 T cells play an important role in controlling viral infections. We investigated the in situ localization of simian immunodeficiency virus (SIV)-specific T cells in lymph and genital tissues from SIV-infected macaques using MHC-class I tetramers. The majority of tetramer-binding cells localized in T cell zones and were CD8⁺. Curiously, small subpopulations of tetramer-binding cells that had little to no surface CD8 were detected in situ both early and late post-infection, and in both vaginally and rectally inoculated macaques. These tetramer⁺CD8^low/− cells were more often localized in apparent B cell follicles relative to T cell zones and more often found near or within the genital epithelium than the submucosa. Cells analyzed by flow cytometry showed similar populations of cells. Further immunohistological characterization revealed small populations of tetramer⁺CD20⁻ cells inside B cell follicles and that tetramer⁺ cells did not stain with γδ-TCR nor CD4 antibodies. Negative control tetramer staining indicated that tetramer⁺CD8^low/− cells were not likely NK cells non-specifically binding to MHC tetramers. These findings have important implications for SIV-specific and other antigen-specific T cell function in these specific tissue locations, and suggest a model in which antigen-specific CD8+ T cells down modulate CD8 upon entering B cell follicles or the epithelial layer of tissues, or alternatively a model in which only antigen-specific CD8 T cells that down-modulate CD8 can enter B cell follicles or the epithelium.     

Allelic Polymorphism,Gene Duplication and Balancing Selection of MHC Class ⅡB Genes in the Omei Treefrog(Rhacophorus omeimontis)

The worldwide declines in amphibian populations have largely been caused by infectious fungi and bacteria. Given that vertebrate immunity against these extracellular pathogens is primarily functioned by the major histocompatibility complex(MHC) class Ⅱ molecules, the characterization and the evolution of amphibian MHC class Ⅱ genes have attracted increasing attention. The polymorphism of MHC class Ⅱ genes was found to be correlated with susceptibility to fungal pathogens in many amphibian species, suggesting the importance of studies on MHC class Ⅱ genes for amphibians. However, such studies on MHC class Ⅱ gene evolution have rarely been conducted on amphibians in China. In this study, we chose Omei treefrog(Rhacophorus omeimontis), which lived moist environments easy for breeding bacteria, to study the polymorphism of its MHC class Ⅱ genes and the underlying evolutionary mechanisms. We amplified the entire MHC class ⅡB exon 2 sequence in the R. omeimontis using newly designed primers. We detected 102 putative alleles in 146 individuals. The number of alleles per individual ranged from one to seven, indicating that there are at least four loci containing MHC class ⅡB genes in R. omeimontis. The allelic polymorphism estimated from the 102 alleles in R. omeimontis was not high compared to that estimated in other anuran species. No significant gene recombination was detected in the 102 MHC class ⅡB exon 2 sequences. In contrast, both gene duplication and balancing selection greatly contributed to the variability in MHC class ⅡB exon 2 sequences of R. omeimontis. This study lays the groundwork for the future researches to comprehensively analyze the evolution of amphibian MHC genes and to assess the role of MHC gene polymorphisms in resistance against extracellular pathogens for amphibians in China.     

Low-Protein Diet Induces IRE1α-Dependent Anticancer Immunosurveillance

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乌鳢(Channa argus)MHC Class I全长cDNA序列的克隆及表达特征

旨在探究乌鳢(Channa argus)MHC基因的分子特征、表达方式及多态性。应用抑制差减杂交(SHH)和快速扩增c DNA末端(RACE)技术克隆并鉴定了乌鳢全长MHC I c DNA序列Char-Ia-1、Char-Ia-2和Char-Ib,推测氨基酸序列与已知硬骨鱼MHC I基因同源。Char-Ia-1和Char-Ia-2 c DNA序列包含1 167和1 083 bp的开放阅读框,分别编码388和360 aa的膜型Ⅰ类分子;而Char-Ib c DNA序列包含978 bp的开放阅读框,编码325 aa,显著截短的羧基末端显示Char-Ib为潜在的分泌型Ⅰ类分子。比较发现Char-Ia与Char-Ib在3'非转录区存在显著差异,在细胞外区、跨膜区和细胞质区的氨基酸序列同源性均较低,推测二者来自不同的MHC I基因座。氨基酸序列比对显示乌鳢MHC I分子与抗原肽结合的关键氨基酸残基较保守,在α1和α3结构域均出现硬骨鱼特征性的氨基酸残基缺失。进化树分析表明Char-Ia-1和Char-Ia-2聚为一簇,与Char-Ib处于不同的进化分支上,进一步证实Char-Ia与Char-Ib分别由不同MHC I基因座编码。RT-PCR分析显示乌鳢MHC I在组织中呈现组成型表达。设计基因专一性引物检测乌鳢Char-Ia与Char-Ib两类MHC I基因在组织中的表达水平,结果显示Char-Ia以较低的浓度表达于所有被检测组织,而Char-Ib主要表达于脾、肠、鳃和外周血,呈现明显的组织表达特异性,提示两类MHC I分子在鱼类免疫反应中发挥不同的生理功能。