五指山小型猪SLA-DQA基因的SNPs及猪人MHC Ⅰ类区自然杀伤细胞KIRs结合区氨基酸变异的比较分析

发布时间：2018-02-01 21:47

  本文关键词： 五指山猪 SLA复合体 单核苷酸多态性 氨基酸变异　出处：《华中农业大学》2005年硕士论文　论文类型：学位论文

【摘要】：异种器官移植一直被认为是解决人体移植器官供体匮乏问题的方法。随着转基因及基因双敲除猪的相继问世,对异种移植的发展产生了巨大的推动作用。MHC编码移植抗原,控制移植排斥反应,参与免疫应答调控和免疫识别,是移植物在受体内能否成活的关键。鉴于此,本实验首次比较详细的完成了五指山小型猪群体中几个经典的SLA基因的SNPs检测,并对五指山小型猪—人异种移植免疫进行初步的研究,获得的结果如下: (1)采用PCR-SSCP结合克隆测序技术,检测五指山猪群体中SLA-DQA,DQB第二外显子的多态性。结果发现SLA-DQA第二外显子存在碱基的颠换(A/C),引起氨基酸的改变(K/Q);而SLA-DQB第二外显子区域没有发现多态。 (2)利用AS-PCR技术对SLA—DQA进行准确分型。设计等位基因序列特异性引物直接从DNA水平上对存在多态的SLA-DQA基因进行分型,并取得了满意的效果,与测序结果100%相符。 (3)依据猪EST所构建的EST重叠群,设计的猪特异引物分离并鉴定五指山猪群体中SLA-1,SLA-2基因第二和第三外显子的多态性。在五指山猪群体中SLA-1,SLA-2基因分别存在两个等位基因,并且这两个基因多肽结合区与人相应部分氨基酸序列间均存在50%～55%的同源性。 (4)对五指山猪群体中SLA Ⅰ类基因核苷酸序列系统数分析,发现与SLA-3基因相比,SLA-1、SLA-2基因间存在较高的同源性。 (5)将猪SLA Ⅰ类基因与人HLA Ⅰ类基因NK细胞杀伤抑制受体结合区相应氨基酸进行了比较分析,结果发现它们都存在一个以上氨基酸的差异,这使得它们与NK细胞杀伤抑制受体基本上很难结合。
[Abstract]:Xenotransplantation has been regarded as a solution to the problem of donor shortage in human organ transplantation. With the development of transgenic and double-knockout pigs. For the development of xenotransplantation, MHC coding for the development of transplantation antigen, control transplant rejection, participate in the regulation of immune response and immune recognition. It is the key to the survival of grafts in vivo. In view of this, the SNPs detection of several classical SLA genes in Wuzhishan mini-pig population was completed in detail for the first time. A preliminary study on the xenotransplantation immunity of Wuzhishan mini-pig and human was carried out, and the results were as follows: 1) SLA-DQA in Wuzhishan pig population was detected by PCR-SSCP combined with cloning and sequencing. The polymorphism of exon 2 of DQB. It was found that the base transversion of exon 2 of SLA-DQA resulted in the change of amino acid. No polymorphism was found in the second exon of SLA-DQB. 2). AS-PCR technique was used to accurately type SLA-DQA. Allelic sequence specific primers were designed to type the polymorphic SLA-DQA gene directly from the DNA level. Satisfactory results were obtained, which were consistent with the sequencing results of 100%. According to the EST overlap population constructed by pig EST, the designed porcine specific primers were used to isolate and identify SLA-1 in Wuzhishan pig population. The polymorphism of exon 2 and exon 3 of SLA-2 gene. There were two alleles in SLA-1 and SLA-2 gene in Wuzhishan pig population. There was a 50% homology between the two polypeptide binding regions and the corresponding amino acid sequences of human. (4) the nucleotide sequence of SLA class I gene in Wuzhishan pig population was analyzed and compared with that of SLA-3 gene. There is high homology among SLA-2 genes. (5) the amino acids in the cytotoxic receptor binding region of porcine SLA class 鈪，

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