Multiplex MHC Typing of Rhesus Macaques

描述(申请人提供)：这项小型研究资助(R03)申请的主要目标是开发一种新的研究技术，能够快速识别猕猴(Macacca Mulatta)基因组中存在的主要组织相容性复合体I类(MHC-I)等位基因。MHC-I基因编码对适应性免疫反应至关重要的蛋白质，通过结合致病性肽并将它们呈递到细胞表面的细胞毒性T细胞来发出感染信号。此外，I类分子对先天免疫反应至关重要，为NK细胞提供抑制和刺激信号。恒河猴是与人类疾病和移植相关的临床前模型，而实验感染的恒河猴是评估艾滋病发病机制、验证治疗方法和制定疫苗接种策略的不可或缺的动物模型。与人类一样，猕猴的病程是不同的，某些MHC-I分子似乎与更好地控制SIV复制有关。目前用于恒河猴基因分型的分子方法使用针对少数Mamu-A和-B等位基因的单独一组序列特异性引物的聚合酶链式反应技术。为了开发一种可以同时检测大量Mamu-A和-B等位基因的DNA检测方法，我们将结合Luminex 100平台的多重功能和含有微小沟槽结合蛋白(MGB)的序列特异性DNA探针(SSP)的高度特异性。MGB允许设计具有更高灵敏度的短探针来区分单核苷酸多态(SNP)，而Luminex微球允许同时使用多达100种不同的探针。我们设计了扩增所有已知的Mamu-A和-B等位基因的外显子2和3的引物。我们将用涂有等位基因特异性探针的Luminex微球来探测这些DNA片段。在猕猴中快速鉴定这些Mamu-A和-B等位基因将对理解MHC-I基因在移植、疫苗开发和使用这一重要动物模型的传染病研究中的作用具有重要意义。

DESCRIPTION (provided by applicant): The main goal of this small research Grant (RO3) application is to develop a new research technology that will allow the rapid identification of Major Histocompatibility Complex class I (MHC-I) alleles present in the genome of rhesus macaques (Macacca mulatta). MHC-I genes encode proteins central to the adaptive immune response, signaling infection by binding pathogenic peptides and presenting them on the cell surface to cytotoxic T cells. In addition, class I molecules are critical to the innate immune response, providing both inhibitory and stimulating signals to NK cells. Rhesus macaques are relevant preclinical models for human diseases and transplantation, and experimentally infected rhesus monkeys serve as an indispensable animal model to assess the pathogenesis, to validate therapy approaches and to develop vaccination strategies against AIDS. As in humans, the disease course in macaques is variable, and certain MHC-I molecules appear to be associated with better control SIV replication. Current molecular methods for genotyping rhesus monkeys use PCR techniques with individual sets of sequence-specific primers for a handful of Mamu-A and -B alleles. In order to develop a DNA assay that can simultaneously detect a large number of Mamu-A and -B alleles, we will combine the multiplexing capacity of the Luminex 100 platform with the high specificity of sequence-specific DNA probes (SSP) that contain minor groove binders (MGB). MGB allow the design of short probes with increase sensitivity to differentiate single nucleotide polymorphism (SNP), whereas the Luminex microspheres permit the simultaneous use of up to 100 different probes. We have designed primers that amplify the polymorphic exons 2 and 3 of all the known Mamu-A and -B alleles. We will probe these DNA fragments with Luminex microspheres coated with allele-specific probes. The rapid identification of these Mamu-A and -B alleles in Rhesus macaques will have significant consequences in understanding the role of MHC-I genes in transplantation, vaccine development, and infectious diseases studies that use this important animal model.