Reagents for the analyses of CMI in the ferret influenza model.

描述(申请人提供)：雪貂有可能成为流感感染的重要动物模型，因为它们最接近于人类发生的疾病症状和病理。雪貂容易感染人类和甲型禽流感，有可能导致人类大流行。然而，由于家雪貂的基因组尚未测序，用于分析雪貂细胞免疫反应的试剂仅限于抗鼠抗体和抗人抗体的交叉反应。为了产生进行这些研究所需的试剂，我们正在从激活的雪貂外周血单核细胞中分离出逆转录病毒cDNAGFP融合蛋白文库。这些表达膜蛋白的克隆将被测序，并通过与猫和狗基因组的同源性进行鉴定。编码这些蛋白的载体可用于分析雪貂细胞免疫反应的免疫学分析，然后可用亚克隆到质粒疫苗载体中的cDNA亚克隆DNA免疫产生小鼠杂交瘤，所产生的鼠单抗在逆转录病毒转导的人细胞系和雪貂细胞上筛选抗原特异性和亲和力。然后，这些试剂将被用于对雪貂对流感病毒攻击的细胞免疫反应的流式细胞学分析。

DESCRIPTION (provided by applicant): Ferrets have the potential to become an important animal model of influenza infection, as they most closely demonstrate the disease symptoms and pathology that occurs in humans. Ferrets are susceptible to infection with human as well as avian influenza A with the potential to cause human pandemics. However, the availability of reagents with which to analyze cellular immune responses in ferrets is limited to cross-reacting anti- mouse and anti-human antibodies as the genome of the domestic ferret has not yet been sequenced. To generate the reagents necessary to perform these studies we are generating retroviral cDNA-GFP fusion protein libraries from mRNA isolated from activated ferret PBMCs. Those clones that express membrane proteins will be sequenced and identified by homology to the feline and canine genomes. The vectors encoding those proteins of interest for use in immunological assays for analysis of ferret cellular immune responses can then be used to generate murine hybridomas by DNA immunization with the cDNA constructs subcloned into plasmid vaccine vectors, and the resulting murine monoclonal antibodies screened for antigen specificity and affinity on retrovirus transduced human cell lines and ferret cells. These reagents will then be validated for flow cytometryic analyses of the cellular immune responses of ferrets to influenza virus challenge.