THE IDENTIFICATION OF AN ANTI-HIV MECHANISM: A PROTEOMIC BASED APPROACH

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 现在人们承认，仅使用高效抗逆转录病毒疗法(HAART)恢复免疫功能是不完整的。由于HIV-1耐药毒株的出现，需要治疗性免疫战略来加强HAART在HIV疾病治疗中的作用。 我们先前观察到，一种多抗原免疫调节剂，即PAI，由灭活流感疫苗和细菌疫苗组成，可诱导MHC非限制性非细胞溶解抗HIV-1活性。基于我们记录的病毒抑制的初步数据，我们建议检验PAI诱导的抗病毒活性可以通过蛋白质组学方法来差异确定的假设。 为了解决这一假设，我们的具体目标是： 具体目的1：通过双向凝胶电泳法(2D-GE)和图像分析，检测经效应细胞培养上清(PBMC、CD8+和CD8+缺失的T细胞)处理的靶T细胞的细胞内裂解产物的差异蛋白表达。 具体目的#2：通过使用质谱仪(MS)、细胞培养中氨基酸稳定同位素标记(SILAC)分析和数据库搜索，鉴定和比较PAI处理的靶T细胞差异表达的蛋白质。 具体目的#3：通过Western blots和/或qRT-PCR验证质谱学鉴定的差异表达蛋白的特性。 初步研究结果表明，PBMC蛋白对PAI治疗有反应。比较主图谱以评估蛋白质表达的差异。这揭示了PAI处理的PBMC中的47个差异表达点。利用串联质谱仪(MS/MS)对改变后的蛋白质进行分析，进行蛋白质鉴定。通过查询NCBInr蛋白质数据库的MS/MS数据，我们鉴定了11个差异表达的蛋白质点。我们相信，鉴定出的蛋白质将产生PAI-抗HIV-1反应的蛋白质组特征。我们将提供不同细胞类型的PAI治疗的蛋白质组参考图谱，以及它们各自相关的HIV机制，供其他人用于比较研究。

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. It is now accepted that restoration of the immune function using only highly active antiretroviral therapy (HAART) is incomplete. Because of the emergence of drug-resistant strains of HIV-1, therapeutic immunization strategies are needed to reinforce HAART in the treatment of HIV disease. We have previously observed that a polyantigenic immunomodulator, known as PAI, which consists of a mixture of inactivated influenza and bacterial vaccine, induces MHC non restricted non-cytolytic anti-HIV-1 activity. Based on our preliminary data documenting viral suppression, we propose to test the hypothesis that PAI induced antiviral activity can be differentially determined by a proteomic approach. To address this hypothesis, our specific aims are: Specific Aim #1: To determine differential protein expression in intracellular lysates from target T cells treated with supernatant from effector cells (PBMC, CD8+and CD8+ depleted T cells) by performing two-dimensional gel electrophoresis (2D-GE) and image analysis. Specific Aim #2: To identify and compare differentially expressed proteins from target T cells treated with PAI by performing peptide mass finger printing using mass spectrometry (MS), stable-isotope labeling by amino acids in cell culture (SILAC) analysis and database search. Specific Aim #3: To validate by Western blots and/or qRT-PCR the identity of differentially expressed proteins identified by mass spectrometry. Preliminary findings identified PBMC proteins responsive to PAI treatment. Master maps were compared to assess differences in protein expression. This revealed 47 differentially expressed spots in PAI treated PBMC. The altered proteins were analyzed by tandem MS (MS/MS) for protein identification. After querying the MS/MS data against the NCBInr protein database, we have identified 11 differentially expressed protein spots. We believe that the identified proteins will generate a proteomic signature of the PAI-anti HIV-1 response. We will make the proteome reference map of PAI treatment in different cell type with their respective related HIV mechanisms available for others to use for comparative studies.