IDENTIFICATION OF CD8+ CELL ANTI HIV FACTOR

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。感染艾滋病毒并保持健康10年以上的个人被视为长期幸存者。这些无症状的个体有很强的细胞介导的免疫反应，抑制HIV复制。当感染者发展成疾病时，这种活动就消失了。抗病毒反应是由分泌的CD8+细胞抗病毒因子（CAF）介导的，该因子通过HIV启动子阻断转录。CAF由来自这些健康感染者的CD8+细胞以低水平产生。这种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鉴定最有可能通过蛋白质纯化和质谱法来实现。方法将健康hiv感染者的CD8+细胞培养在不含血清和白蛋白的培养基中。在实验室用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、色谱和分级柱）对活性流体进行浓缩和分馏。具有富集CAF活性的馏分用二维凝胶分析，并用质谱法评估。到目前为止，我们已经能够证明CAF的分子大小约为50 kD。通过2D凝胶分析，至少有20个不同的点与CAF活性相关。质谱分析的研究正在进行中。这些蛋白质斑点是独特的含钙液体将进一步评估。通过质谱分析，我们希望能够鉴定介导这种抗hiv活性的蛋白质（CAF）。

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. Introduction 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. Methods 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. Results Thus far, we have been able to show that CAF has a molecular size of about 50 kD. By differential 2D gel analysis, there appears to be at least 20 different spots that are associated with CAF activity. Studies are in progress to analyzed by mass spectrometry. Those protein spots that are unique to CAF-containing fluids will be further evaluated. Discussion By mass spectrometry, we hope to be able to identify the protein (CAF) that mediates this anti-HIV activity.