【作者】 戴银；

【导师】 余为一；

【作者基本信息】 安徽农业大学 ， 微生物学， 2010， 博士

【摘要】 主要组织相容性复合体（MHC）由一组紧密连锁、高度多态的基因座所组成,在脊椎动物的移植排斥反应、免疫应答和免疫调控等方面起极其重要的作用。鸡的MHCⅠ基因（BF）的多态性与许多传染病的抗性和易感性密切相关,所以,研究MHC多态性特征是抗病育种的基础。虽然关于禽类MHCⅠ类分子结构特性的研究已有许多报道,但由MHC基因导致不同品种间抗病性差异的分子机理尚不十分清楚。MHC分子的多态性是动物在环境压力下平衡选择的结果。由于家畜禽在饲养选择中更多地受到人工选择,特别是传染病原压力影响,不同区域形成的在外形、生产性能等方面可能完全不同,是否其MHCⅠ分子在结构演变中也产生差异。为此,我们选择了在中国南方和中部地区的三个经过长期驯化的鸡品种（即文昌鸡、清远和淮北麻鸡）,运用RT-PCR法克隆了清远麻鸡、淮北麻鸡和文昌鸡的MHCⅠ类分子cDNA序列,进行了结构分析。首先,经过克隆,从90羽鸡共获得了80条α链cDNA序列,其中文昌鸡、清远和淮北麻鸡分别为24、30和26条。序列分析显示α链呈现高度多态,有73不同基因序列,还存在6组的序列是完全相同的,其中3组的成员分布于不同品种。另,我们测序了15条β_2m链cDNA序列,每个品种各5条,分析发现其序列差异极小,较为保守。进一步分析MHCⅠα氨基酸全长序列位点的分布特征,显示鸡MHCⅠ类分子的多态性氨基酸位点共130个,分布整个分子的5个区域（信号肽、α1、α2、α3和TM / CY区域）,但是其中83个集中在α1和α2区域（9～155aa）。构建了MHCⅠα链序列系统发育树,结果显示克隆的等位基因型与其他鸡品种一起分属于两个类群,决定分属类群的是基因型而不是品种。这表明鸡MHCⅠα具有共同祖先的遗传特征,其品种属性与MHC分属类群无相关性。由此推测,驯化鸡更多地受到共同病原的压力选择,而不是受地域环境影响。其次,采用Wu-kabat法分析了α链α1和α2区域位点的变异率,并与其他物种进行了α1和α2区域多态位点和空间结构的比对。分析多态性位点发现,清远麻鸡的α1和α2区域氨基酸序列共有16处高度多态性位点（分值大于等于4）,其中α1区域有9个,α2区域有7个,而这两个区的4个高峰的位点分布于9、111、113和153位（分值大于等于8）;淮北麻鸡在α1和α2区域共23个高度变异位点,分别为10和13个,类似清远鸡,位点9、111、113和153的变异值较高,特别是113变异率高达26;文昌鸡的α1和α2区域存在25个高度变异位点,分别为13和12个,其中位点9、69、111、113、149和153位为5个峰点。此外,我们发现三个品种MHCⅠ类分子的位点9、24、43、53、72、97、111、113和153都是高度变异,且9、111、113和153变异率分值均大于或等于8。最后,通过对比鸭、人和鼠MHCⅠα的高变异位点,显示禽类的9、111、113和153位氨基酸对应哺乳动物中的9、114、116和156均属高度变异位点。在MHCⅠα链分子中存在抗原肽结合域（PBR）,是MHCⅠ分子多态性最高的区域,也是与抗病性关系最密切的结构。PBR位于α1、α2区,所以不同物种MHCⅠ的α1和α2区域氨基酸残基在分布上存在共同特征,尤其是在PBR中与抗原结合位点的空间定位具有相似性。这些结果表明,脊椎动物MHC分子是物种分离前形成的,具有共同祖先的特征。在进化过程中,同一物种尽管所生存的环境不同,但是主要还是受到病原体选择,在品种间存在更多的共同特征。由于MHCⅠ类分子α和β链在细胞内先形成稳定聚合体,才能具有识别结合抗原短肽功能,进而呈递抗原肽。虽然PBR位于α上,但β链对于MHCⅠ分子形成稳定空间结构和发挥功能是必须的。为了探索MHC呈递处理抗原的机制,本文还构建了重组MHCⅠ类分子αβ链片段,并进行了原核表达。为此,我们利用基因工程技术将MHCⅠ分子的α和β通过一端连接肽头尾相接,构建了pET-MHCⅠβ_2m-α原核表达重组质粒,并转入了大肠杆菌BL21进行了表达。结果表明重组质粒得到了顺利表达,且获得了具有一定活性的MHCⅠβ_2m-α融合蛋白。该研究为进一步探索MHCⅠ分子携带抗原肽机制,保持和促进其功能的稳定发挥提供了实验基础。更多还原

【Abstract】 Major histocompatibility complex （MHC） is closely linked by a group of highly polymorphic loci, they play a crucial role in the immune system, which induce the animal body transplant rejection, present antigen peptides to T cells for recognition, and trigger an appropriate immune response. Polymorphism of chicken MHCⅠgene （BF） is associated with resistance and susceptibility of many important infectious diseases, so the knowledge about characters of MHC polymorphism is basic for resistibility breeding. A number of works focusing on poultry MHCⅠgene have been reported; still it is not clear about the molecular mechanism of MHC inducing diversity in resistance to infectious diseases between animal breeds. MHC polymorphisms are subject to the animal’s response to the environmental pressure. Livestock and poultry in the breeding process are more artificially selected and bred, especially under infectious pathogen pressure, which inhabit in different areas and exhibit various produce and form characters, whether their MHC gene has diversity in evolvement under the pressure. In order to understand polymorphism character of MHC evolution in domesticated poultry we selected three local breeds of chicken （Qingyuan, Wenchang and Huaibei） in different areas of China, and cloned and sequenced MHCⅠcDNA gene.First 80 sequences of MHCⅠαgene were cloned from 90 individuals by RT-PCR, in which Qingyuan, Wenchang and Huaibei were 30, 24 and 26 respectively. Comparison of sequences indicated thatαgene exhibited high polymorphism and had 73 different sequences and 6 groups having identical sequence, in which there were 3 groups, its members were distributed in different breeds. We also sequenced 5β_2m gene, they exhibited high conversed and less diversity. Further we analyzed the distribution of polymorphic amino acid residues in chicken MHCⅠαchain, in which an aligning all sequences of amino acid the residues. The results revealed that there were 130 of polymorphic sites, which were distributed in total MHCⅠαchain including five regions, namely leader peptide,α1,α2,α3 and TM / CY regions, but 83 of them were located in theα1 andα2 regions （9¹55aa）.In the phylogenetic tree all alleles belonged to two clusters together with other different taxa, especially one individual as cluster member was dependent on its genotype rather breed. This suggest that no correlation of MHCⅠαwith the chicken breeds and they keep genetic characters of a common ancestor. It is presumed that the MHCⅠmolecule of domesticated chickens would more influenced by the pressure of the pathogens rather than the environment in different areas.Secondly, analysis and comparison of diversity inα1 andα2 regions with other species were carried out by Wu-kabat index and structure simulation. We found that 16 amino acid residues with high polymorphism （score≥4） in PBRs of Qingyuan Partridge chicken MHCⅠ. There were 9 of polymorphic sites inα1 region, 7 of those sites inα2 region. Four main peaks （score≥8） were located at the residues 9, 111, 113 and 153. A total of 23 polymorphic sites in Huaibei Partridge chicken MHCⅠwere located in the regions, including 10 sites ofα1 region , 13 sites ofα2 region, four main peaks were also located at the residues 9, 111, 113 and 153. Wenchang chicken MHCⅠhad 25 highly variable sites,α1 andα2 regions had 13 and 12 sites respectively, and the residues 9,69,111,113,149 and153 with a score were greater than or equal to 8. Moreover, all these residues 9, 69, 111, 113, 149 and153 were polymorphic in three breeds, and the sites 9, 111, 113 and153 were highly polymorphic （score≥8）. Compared with duck, human and mouse, the distribution of amino acid residues inα1 andα2 regions ofα-chain shows some similar features, namely some variable polymorphic sites （score≥4） at 9, 111（114）, 113 （116） and 153 （156）, especially these polymorphic sites in peptide-binding regions （PBRs） and their spatial location, indicating that they had similarity in gene structure. All these results suggest that MHC gene is an ancestral sequences that existed before separation of specie and has common character. In the evolution process the MHC gene of breeds in same specie have keep more common characters because under pressure of the specific pathogen rather than different living environment.A presenting antigen peptide is dependent on polymerization between MHCαandβchain and forming specific stable structure. The PBR locates inαchain, butβ_2m is essential for MHCⅠmolecule function. To explore mechanism of MHC molecule presenting antigen we constructed a recombined segment containing MHCⅠαandβchains and expressed in a prokaryotic system. We connectedαchain toβchain with a linker and inserted it into a vector, this recombinant plasmid was named as pET-MHCⅠβ_2m-α. Then it was transferred into E. coli BL21 and expressed. The results showed that the pET-MHCⅠβ_2m-αwere successfully expressed, This MHCⅠβ_2m-αfusion protein with certain immune activity would provide experimental materials for exploring mechanism of MHCⅠmolecule improving immune function as epitope carrier.更多还原