Department of Microbiology and Immunology, University of Melbourne, Parkville, Vic, Australia.
Immunogenetics. 2011 Aug;63(8):511-21. doi: 10.1007/s00251-011-0529-5. Epub 2011 May 10.
Pig-tailed macaques (Macaca nemestrina) are a commonly studied primate model of human AIDS. The Mane-A1084:01 MHC class I allele (previously named Mane-A10) is important for the control of SIV infection by CD8+ T cells in this model. Validated methods to detect this allele in large numbers of macaques are lacking. We studied this MHC allele using sequence-specific PCRs in 217 pig-tailed macaques and identified 75 (35%) positive animals. We then performed massively parallel pyrosequencing with a universal 568-bp MHC class I cDNA-PCR amplicon for 50 of these 75 macaques. All 50 animals expressed Mane-A1084:01 or closely related variants of the Mane-A1084 lineage. Mane-A1084 transcripts accounted for an average of 20.9% of all class I sequences identified per animal. SIV infection of a subset of these macaques resulted in the induction of SIV-specific CD8+ T cell responses detected by Mane-A1084:01 tetramers. An average of 19 distinct class I transcripts were identified per animal by pyrosequencing. This analysis revealed 89 new Mane class I sequences as well as 32 previously described sequences that were extended with the longer amplicons employed in the current study. In addition, multiple Mane class I haplotypes that had been inferred previously based on shared transcript profiles between unrelated animals were confirmed for a subset of animals where pedigree information was available. We conclude that sequence-specific PCR is useful to screen pig-tailed macaques for Mane-A1*084:01, although pyrosequencing permits a much broader identification of the repertoire of MHC class I sequences and haplotypes expressed by individual animals.
猪尾猕猴（Macaca nemestrina）是一种常用于研究人类艾滋病的灵长类动物模型。在该模型中，Mane-A1084:01 MHC Ⅰ类等位基因（以前称为 Mane-A10）对 CD8+T 细胞控制 SIV 感染非常重要。目前缺乏用于在大量猕猴中检测该等位基因的验证方法。我们使用序列特异性 PCR 在 217 只猪尾猕猴中研究了这个 MHC 等位基因，发现 75 只（35%）阳性动物。然后，我们对这 75 只猕猴中的 50 只进行了大规模平行焦磷酸测序，使用的是一个通用的 568bp MHC Ⅰ类 cDNA-PCR 扩增子。这 50 只动物都表达了 Mane-A1084:01 或与 Mane-A1084 谱系密切相关的变体。Mane-A1084 转录本平均占每只动物鉴定的所有 I 类序列的 20.9%。对其中一部分猕猴进行 SIV 感染后，诱导了 Mane-A1084:01 四聚体检测到的 SIV 特异性 CD8+T 细胞反应。焦磷酸测序平均每只动物鉴定到 19 个不同的 I 类转录本。该分析揭示了 89 个新的 Mane Ⅰ类序列以及 32 个以前描述的序列，这些序列在当前研究中使用的较长扩增子进行了扩展。此外，根据亲缘关系较远的动物之间共享的转录谱推断出的多个 Mane Ⅰ类单倍型，在可获得系谱信息的动物亚群中得到了证实。我们得出结论，序列特异性 PCR 可用于筛选猪尾猕猴的 Mane-A1*084:01，尽管焦磷酸测序可以更广泛地鉴定个体动物表达的 MHC Ⅰ类序列和单倍型。
