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

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的有力候选者的角度提供重要信息。在此，我们对SLA I类区域400kb片段进行了大规模基因组测序，并设计了用于SLA I类基因遗传多态性分析的位点特异性引物对，在该片段中发现了19个与19个人类非MHC基因对应的猪同源基因。这些基因的基因序列在人和猪之间高度保守。相反，在HSR1和Flj2 2638基因之间没有识别出SLA基因，而在相应的人类片段中存在HLA-E基因。包括这一片段的SLA I类区域的长度仅为979 kb，而人类白细胞抗原I类区域的长度长达1550 kb。这种显著的大小差异可以用人类MHC中较高密度的重复序列来解释。此外，我们设计了8对位点特异性引物对表达的三个经典SLA I类基因SLA-1、SLA-2和SLA-3的遗传多态性进行了RT-PCR分析。对RT-PCR产物的序列分析已经能够鉴定先前由PD1、PD14、PCIcDNA序列和SLA-3基因组序列定义的等位基因。在SLA-1、SLA-2和SLA-3基因中也发现了新的等位基因。此外，我们还设计了8对等位基因特异性引物。用该套引物对克劳恩小型猪的5个SLA I类和2个DRB1等位基因进行了精确的PCR-SSP分析。该方法操作简便、快速，可用于SLA纯合子克劳恩小型猪的SLA基因分型。

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.