IDENTIFICATION OF CD8+ CELL ANTI HIV FACTOR

CD8细胞抗HIV因子的鉴定

• 批准号: 7724166
• 负责人: JAY A LEVY
• 金额: $ 1.15万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7724166
• 关键词: Albumins; Antiviral Agents; Antiviral Response; Biochemical; Biological Assay; CD8-Positive T-Lymphocytes; Cells; Column Chromatography; Computer Retrieval of Information on Scientific Projects Database; Culture Media; Disease; Funding; Gel; Genetic Transcription; Grant; Growth Factor; HIV; Human; Individual; Institution; Ion-Exchange Chromatography Procedure; Isotope Labeling; Laboratories; Liquid substance; Long-Term Survivors; Mass Spectrum Analysis; Mediating; Persons; Procedures; Proteins; Research; Research Personnel; Resources; Serum; Source; Standards of Weights and Measures; Techniques; United States National Institutes of Health; cell mediated immune response; chemokine; cytokine; numb protein; promoter; protein purification; research study; size

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Individuals who have been infected with HIV and remain healthy for over 10 years are considered long-term survivors. These asymptomatic individuals have a strong cell-mediated immune response that suppresses HIV replication. This activity is lost as the infected person advances to disease. The antiviral response is mediated by a secreted CD8+ cell antiviral factor (CAF) which blocks transcription via the HIV promoter. CAF is produced at low levels by CD8+ cells from these healthy infected individuals. The protein appears to be unlike any of the known cytokines, chemokines and human growth factors. CAF identification will most likely be achieved through protein purification and mass spectrometry. CD8+ cells from healthy HIV-infected individuals are grown in culture medium lacking serum and where possible, albumin. Fluids are assayed for antiviral activity by standard procedures in the laboratory. Active fluids are then concentrated and fractionated by various biochemical techniques including ion exchange, chromatography, and by sizing columns Fractions with enriched CAF activity are analyzed by 2-D gel and evaluated by mass spectrometry. By column chromatography, we have been able to isolate two fractions from a sizing column (TSK) which have anti-HIV activity. Each of them has a limited number of proteins, which are being further evaluated as possible candidates for CAF activity. We are conducting isotope labeling experiments in attempts to quantitate differences in proteins expressed in CAF+ and CAF- fluids. By further mass spectrometry, we hope to be able to identify the protein (CAF) that mediates this anti-HIV activity.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 感染艾滋病毒并保持健康超过 10 年的个人被视为长期幸存者。 这些无症状个体具有强烈的细胞介导的免疫反应，可抑制艾滋病毒复制。 随着感染者患病，这种活性就会消失。 抗病毒反应由分泌的 CD8+ 细胞抗病毒因子 (CAF) 介导，该因子通过 HIV 启动子阻断转录。 来自这些健康感染者的 CD8+ 细胞会产生低水平的 CAF。 该蛋白质似乎不同于任何已知的细胞因子、趋化因子和人类生长因子。 CAF 鉴定很可能通过蛋白质纯化和质谱来实现。 来自健康 HIV 感染者的 CD8+ 细胞在缺乏血清和（可能的话）白蛋白的培养基中生长。 在实验室中通过标准程序测定液体的抗病毒活性。 然后通过各种生化技术（包括离子交换、色谱法和分级柱）对活性流体进行浓缩和分级。通过 2-D 凝胶分析具有富集 CAF 活性的级分，并通过质谱法进行评估。 通过柱色谱，我们已经能够从分级柱 (TSK) 中分离出两种具有抗 HIV 活性的组分。 它们各自的蛋白质数量有限，正在进一步评估这些蛋白质是否具有 CAF 活性。 我们正在进行同位素标记实验，试图定量 CAF+ 和 CAF- 液体中表达的蛋白质的差异。 通过进一步的质谱分析，我们希望能够鉴定出介导这种抗 HIV 活性的蛋白质 (CAF)。