Molecular Biology Core

分子生物学核心

• 批准号: 8376704
• 负责人: AYMERIC DE PARSEVAL
• 金额: $ 37.9万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8376704
• 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; polyclonal antibody; 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协助HIVRAD计划确定表位 被靶向QNE的MAb和多克隆人和猴抗血清识别。这其中的主要作用 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 核心本研究的具体目的是：1）设计并制备表达嵌合型和突变型HIV-1的质粒 项目1、2和4中表位作图研究和病毒中和试验的Envs。2)完成网站 单体WT gp 120和gp 140蛋白，以及表达QNE的可溶性单体和三聚体。这些 蛋白质将用于项目1、2和4中的结合和吸附研究，并在项目2中用作免疫原。 3.这些研究将由从项目1、2和3获得的表位作图实验的结果指导。 4. 3)为了产生感染性SHIV假型和感染性突变体SHIV， QNE。将SHIV Env的片段克隆到合适的载体中以促进诱变 将完整的SHIV Env-Rev基因亚克隆到pcDNA 3.1载体中， 产生病毒假型。这些将用于允许Env突变体和嵌合体的有效构建 以及用于产生病毒假型以表征突变体SHIV的功能活性 环境项目1和2中定义的QNE将被引入SHIV 1157 ip环境中，以表征 它们在猴血PBMC中的感染性和它们的中和敏感性。最终，突变的SHIV Envs 引入SHIV 1157 ip前病毒骨架以产生表达体内QNE的感染性SHIV 项目3中描述的研究。4)从感染的大肠杆菌中分离和鉴定逃逸突变体Envs， 猕猴在感染动物中Env序列的演变将在研究过程中进行研究。 感染，以跟踪抗QNE中和反应并鉴定病毒逃逸突变体。Envs将是 克隆和逃逸突变体将通过中和试验（Viral Immunology core）鉴定。