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

描述（由申请方提供）：雪貂有可能成为流感感染的重要动物模型，因为它们最接近地展示了人类中发生的疾病症状和病理。雪貂容易感染人类和禽流感A，有可能引起人类大流行。然而，用于分析雪貂中细胞免疫应答的试剂的可用性限于交叉反应的抗小鼠和抗人抗体，因为家养雪貂的基因组尚未测序。为了产生进行这些研究所需的试剂，我们从从活化的雪貂PBMC分离的mRNA产生逆转录病毒cDNA-GFP融合蛋白文库。将对表达膜蛋白的那些克隆进行测序，并通过与猫和犬基因组的同源性进行鉴定。然后，编码那些用于分析雪貂细胞免疫应答的免疫学测定的目的蛋白的载体可用于通过用亚克隆到质粒疫苗载体中的cDNA构建体进行DNA免疫来产生鼠杂交瘤，并筛选所得鼠单克隆抗体对逆转录病毒转导的人细胞系和雪貂细胞的抗原特异性和亲和力。然后将验证这些试剂用于白鼬对流感病毒攻击的细胞免疫应答的流式细胞术分析。 公共卫生相关性：完成拟议的研究将提供对甲型流感感染的细胞免疫反应进行分析的能力，并确定雪貂对这种病毒的免疫保护的相关性。此外，cDNA文库将有助于那些有兴趣在淋巴组织和PBMC中的雪貂基因表达的进一步研究。类似的方法将可用于产生用于基因组序列数据不可用的其他模式生物（例如棉鼠）的抗体试剂。

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. PUBLIC HEALTH RELEVANCE: Completion of the proposed research will provide the ability to perform analyses of cellular immune responses against influenza A infection and determine the correlates of immune protection against this virus in ferrets. In addition, the cDNA libraries will be useful for those interested in further studies of ferret gene expression in lymphoid tissues and PBMCs. Similar methodology would be useful for generating antibody reagents for other model organisms for which genomic sequence data is not available, such as the cotton rat.