Identification of DRB alleles in rhesus monkeys using polymerase chain reaction-sequence-specific primers (PCR-SSP) amplification
Identification of DRB alleles in rhesus monkeys using polymerase chain reaction-sequence-specific primers (PCR-SSP) amplification
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DOI：
10.1034/j.1399-0039.1999.540306.x
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发表时间：
1999-09-01
期刊：
影响因子：
--
通讯作者：
Thomas, J
中科院分区：
文献类型：
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作者：
Lobashevsky, A;Smith, JP;Thomas, J
主要组织相容性复合体(MHC)II类分子在哺乳动物免疫系统中对T细胞功能的调节起着至关重要的作用。MHC单倍型在人类和非人类灵长类动物中的多态性有两个关键特征：大量等位基因的存在，以及这些等位基因之间高度的遗传多样性。恒河猴和黑猩猩已被广泛用作人类疾病和移植的相关模型。我们用序列特异性引物聚合酶链式反应(PCR-SSP)和变性梯度凝胶电泳法(DGGE)对3个科19只猕猴的DRB基因进行了研究。扩增后，对扩增产物进行纯化和测序。7只动物(麦迪逊1号)也被DDGE分型。我们报道，由PCR-SSP定义的DRB单倍型与DGGE和直接测序获得的数据高度一致。我们的数据显示，在这些家系中，DRB1等位基因的数量存在显著的差异，从id到每个基因型，这一分析表明，大多数扩增片段与我们的聚合酶链式反应引物要扩增的Mamu-DRB等位基因相同。而MAMU-DRB1*0303、MAMU-DRB6*0103和MAMU-DRB*W201等位基因的相似性为98-99%。观察到的错配位于非多态区域。因此，用分子方法对猕猴进行的家系研究证实了每个单倍型的Mamu-DRB1等位基因的多样性和早些时候发表的等位基因关联的存在。此外，我们还提出了3个DRB等位基因关联(单倍型)：MAMU-DRB1*04-DRB5*03；MAMU-DRB1*04-*DRB*W5；MAMU-DRB1*04*W2。所提出的中等分辨率的PCR-SSP技术似乎是一种检测恒河猴DRB基因多态的高度重复性和区分性的分型方法。
Major histocompatibility complex (MHC) class II molecules play a vital role in the regulation of T-cell functions in the mammalian immune system. Two key features characterize the polymorphism of MHC haplotypes in humans and non-human primates: the existence of a large number of alleles, and the high degree of genetic diversity between those alleles. Rhesus monkeys and Chimpanzees have been extensively used as relevant models for human diseases and transplantation We have investigated DRB genes in 19 macaques, members of 3 families, using polymerase chain reaction with sequence-specific primers (PCR-SSP) and denaturing gradient gel electrophoresis (DGGE). After amplification PCR products were purified and subjected direct sequencing. Seven animals (Madison #1) were typed by DDGE also. We report that the DRB haplotypes defined by PCR-SSP exhibit a high degree of concordance with the data obtained by DGGE and direct sequening. Our data show prominent variability in the number of DRB1 alleles ranging from id, per genotype within these families, This analysis demonstrated that most of the amplicons were identical to Mamu-DRB alleles that our PCR primers were to amplify. However, 98-99% similarity was noticed in the case of Mamu-DRB1*0303, Mamu-DRB6*0103 and Mamu-DRB*W201 alleles. The observed mismatches were located in non polymorphic regions. Thus, family studies in rhesus macaques performed by molecular methods confirmed the multiplicity of Mamu-DRB1 alleles per haplotype and the existence of allelic associations published earlier. In addition, we propose 3 more DRB allele associations (haplotypes): Mamu-DRB1*04-DRB5*03; Mamu-DRB1*04-*DRB*W5; Mamu-DRB1*04*W2. The proposed medium-resolution PCR-SSP technique appears to be a highly reproducible and discriminatory typing method for detecting polymorphisms of DRB genes in rhesus monkeys.