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

用于雪貂流感模型中 CMI 分析的试剂。

基本信息

批准号：
8328600
负责人：
KAREN Marie DUUS
金额：
$ 7.9万
依托单位：
ALBANY MEDICAL COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-15 至 2013-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8328600
关键词：
Affinity Animal Model Antibodies Antigens Avian Influenza B-Lymphocytes Binding Biological Assay CD19 gene CD28 gene CD3 Antigens CD44 gene CD8B1 gene Canis familiaris Cell Lineage Cell membrane Cells Chimeric Proteins Collaborations Complementary DNA Cotton Rats DNA Data Development Disease Family Felidae Ferrets Flow Cytometry Gene Expression Gene Expression Profile Gene Targeting Generations Genes Genome Genomics Goals Human Human Anti-Mouse Antibody Human Cell Line Hybridomas ITGAM gene ITGAX gene Immune Immune Cell Activation Immune response Immunization Immunoassay Infection Influenza Influenza A virus Interferons Interleukin-2 Interleukin-4 Laboratories Lentivirus Vector Libraries Lymphoid Tissue Mediating Membrane Membrane Proteins Messenger RNA Methodology Modeling Mono-S Monoclonal Antibodies Mus Pathology Plasmids Population Proteins Reagent Research Retroviridae SELL gene Screening procedure Sorting - Cell Movement Specificity Symptoms T-Lymphocyte TNF gene TNFRSF5 gene TNFSF5 gene Testing Vaccination Validation Virus base cDNA Library cell mediated immune response cellular transduction cytokine genome sequencing granzyme B influenza virus vaccine influenzavirus interest meetings pandemic disease particle vector vector vaccine

项目摘要

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.

描述（由申请人提供）：雪貂有可能成为流感感染的重要动物模型，因为它们最接近人类的疾病症状和病理。雪貂容易感染人类和禽流感，有可能引起人类大流行。然而，用于分析雪貂细胞免疫反应的试剂的可用性仅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。为了生成进行这些研究所需的试剂，我们正在从活化的雪貂pbmc中分离的mRNA中生成逆转录病毒cDNA-GFP融合蛋白文库。这些表达膜蛋白的克隆将通过与猫和犬基因组的同源性进行测序和鉴定。编码这些蛋白的载体可用于分析雪貂细胞免疫应答的免疫学试验，然后将cDNA构建体亚克隆到质粒疫苗载体中，通过DNA免疫产生小鼠杂交瘤，并筛选产生的小鼠单克隆抗体对逆转录病毒转导的人类细胞系和雪貂细胞的抗原特异性和亲和力。然后，这些试剂将被验证用于雪貂对流感病毒攻击的细胞免疫反应的流式细胞术分析。