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Genome analysis of the swine MHC region and development of SLA-DNA typing method for xenotransplantaion

猪MHC区域的基因组分析和异种移植SLA-DNA分型方法的开发

基本信息

批准号：
13671256
负责人：
ANDO Asako
金额：
$ 2.24万
依托单位：
Tokai University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671256/
关键词：
xenotransolantation miniature swine MHC SLA region HLA region genome analysis SLA gene DNA typing ゲノム構造

项目摘要

Genome analysis on the swine leukocyte antigen (SLA) region and analysis of genetic polymorphisms of the SLA genes will give important information from a viewpoint of a possible strong candidate of pig organs as a donor for xenotransplantation. Here, we carried out large scale genome sequencing of the 400 kb segment in the SLA class I region and designed locus-specific primer pairs for analysis of genetic polymorphisms of the SLA class I genes.Nineteen swine homologous genes corresponding to nineteen human non-MHC genes were identified in this segment in the SLA class I region. The gene order of these genes was highly conserved between the human and the swine. In contrast, no SLA gene was recognized between the HSR1 and FLJ22638 genes, as compared with the presence of the HLA-E gene in the corresponding human segment. The length of the SLA class I region including this segment was only 979 kb, as contrasted with as long as 1550 kb in the HLA class I region. This remarkable difference in size between them could be explained by a higher density of repetitive sequences in the human MHC.Furthermore, we designed eight locus-specific primer pairs for RT-PCR analyses of genetic polymorphisms of the expressed three classical SLA class I genes, SLA-1, SLA-2 and SLA-3. Sequence analysis of the RT-PCR products has allowed the identification of alleles which were previously defined by the PD1, PD14, PCI cDNA sequences, and SLA-3 genomic sequence. Novel alleles were also defined in the SLA-1, -2, and -3 genes.In addition, we also designed eight allele-specific primer pairs for the PCR-SSP method. These primer sets have allowed to assign five SLA class I and two DRB1 alleles of Clawn miniature swine precisely by the PCR-SSP method. The rapid and simple processing of the PCR-SSP method will be useful for SLA genotyping in selection of SLA-homozygous Clawn miniature swine.

对猪白细胞抗原（SLA）区域的基因组分析和SLA基因的遗传多态性分析将从猪器官作为异种移植供体的可能性强的候选者的观点提供重要信息。本研究对SLA I类基因区400 kb片段进行了大规模基因组测序，并设计了位点特异性引物，对SLA I类基因的遗传多态性进行了分析，在该片段中发现了19个猪的同源基因，对应于19个人的非MHC基因。这些基因的基因顺序在人和猪之间高度保守。相反，与HLA-E基因存在于相应的人片段中相比，在HSR1和FLJ22638基因之间没有识别出SLA基因。包括该片段的SLA I类区域的长度仅为979 kb，而HLA I类区域的长度则高达1550 kb。此外，我们设计了8对位点特异性引物，对SLA-1、SLA-2和SLA-3这3个经典的SLA I类基因进行了RT-PCR分析。RT-PCR产物的序列分析允许鉴定先前由PD1、PD14、PCI cDNA序列和SLA-3基因组序列定义的等位基因。此外，我们还设计了8对等位基因特异性引物，用于PCR-SSP方法。利用PCR-SSP方法对克劳恩小型猪的5个SLA Ⅰ类和2个DRB 1等位基因进行了精确的定位。PCR-SSP方法操作简便、快速，可用于SLA纯合型克朗斯小型猪的基因分型。