Molecular Biology Core

分子生物学核心

• 批准号: 7904637
• 负责人: AYMERIC DE PARSEVAL
• 金额: $ 37.65万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7904637
• 关键词: Adsorption; Animals; Antibodies; Antigens; Autologous; Binding; Biological Assay; Blood; Chimera organism; Codon Nucleotides; Collaborations; Consensus; DNA; Data; Epitope Mapping; Epitopes; Escape Mutant; Evolution; Generations; Goals; HIV; HIV Envelope Protein gp120; HIV-1; Human; Immune Sera; Immunology; Infection; Length; Macaca; Molecular; Molecular Biology; Monkeys; Monoclonal Antibodies; Mutagenesis; Patients; Plasma; Plasmids; Play; Protein Binding; Proteins; Reagent; Research Project Grants; Role; Series; Serum; Site; Sorting - Cell Movement; Testing; Time; Variant; Vertebral column; Viral; Virus; Work; base; cell type; design; env Gene Products; env Genes; expression vector; improved; in vivo; interest; monomer; mutant; programs; research study; response; rev Genes; simian human immunodeficiency virus; vector

The purpose of the Molecular Biology Core is to assist the HIVRAD program in characterizing the epitopes recognized by MAbs and polyclonal human and monkey antisera that target QNEs. The main role of this Core will be to generate DNA and protein reagents needed by the other projects and the Viral Immunology Core. The Specific Aims are : 1) To design and produce plasmids expressing chimeric and mutant HIV-1 Envs for epitope mapping studies and for viral neutralization assays in Projects 1, 2 and 4. 2) To generate monomeric WT gp120 and gp140 proteins, and soluble monomers and trimers that express QNEs. These proteins will be used for binding and adsorption studies in Projects 1, 2 and 4, and as immunogens in Project 3. These studies willl be guided by results of epitope mapping experiments obtained from Projects 1, 2 and 4. 3) To generate infectious SHIV pseudotypes and infectious mutant SHIVs with variant Envs that express QNEs. Fragments of the SHIV Env will be cloned into appropriate vectors to facilitate mutagenesis experiments, and the complete SHIV Env-Rev genes will be subcloned into the pcDNA 3.1 vector to generate viral pseudotype. These will be used to allow efficient construction of Env mutants and chimeras and for the generation of viral pseudotypes for the characterization of functional activity of the mutant SHIV Envs. QNEs defined in Projects 1 and 2 will be introduced into the SHIV1157ip Env for characterization of their infectivity in monkey blood PBMCs and their neutralization sensitivity. Ultimately, mutant SHIV Envs will be introduced in the SHIV1157ip proviral backbone to generate infectious SHIVs expressing QNEs for invivo studies described in Project 3. 4) To isolate and characterize escape mutant Envs from infected macaques. The evolution of Env sequence in the infected animals will be investigated during the course of infection to follow the anti-QNEs neutralizing responses and identify viral escape mutants. Envs will be cloned and escape mutants will be identified by neutralization assays (Viral Immunology core).

分子生物学核心的目的是帮助HIVRAD计划表征表位 可被以QNE为靶点的单抗和多克隆人、猴抗血清识别。它的主要作用是 核心将是产生其他项目和病毒免疫学所需的DNA和蛋白质试剂 核心。具体目标是：1)设计和生产表达嵌合和突变的HIV-1的质粒 项目1、2和4)中用于表位映射研究和病毒中和分析的环境 单体WT gp120和gp140蛋白，以及表达QNEs的可溶性单体和三聚体。这些 蛋白质将在项目1、2和4中用于结合和吸附研究，并在项目中用作免疫原 3.这些研究将以项目1、2和2获得的表位映射实验结果为指导 4.3)产生感染性Shiv伪型和具有表达的变异型env的感染性突变体Shiv QNEs。SHIV Env的片段将被克隆到适当的载体中，以促进突变 实验中，将完整的Shiv env-rev基因亚克隆到pcDNA3.1载体中，以 产生病毒假型。这些将被用于有效地构建环境突变体和嵌合体 以及用于鉴定突变体Shiv功能活性的病毒假型的产生 环境。项目1和2中定义的QNE将被引入SHIV1157ip环境中，用于表征 它们对猴血PBMCs的感染性和中和敏感性。最终，突变的Shiv env将 被引入SHIV1157ip前病毒骨架中，产生感染性SHV，表达QNEs用于体内 项目3.4)中描述的从感染中分离和鉴定逃逸突变环境病毒的研究 猕猴。在感染动物的过程中，将研究env序列的演变。 感染后跟踪抗QNEs中和反应，鉴定病毒逃逸突变体。环境将是 克隆和逃逸突变体将通过中和试验(病毒免疫学核心)进行鉴定。