Mass Spectrometric Detection of Drug-Resistant HIV

耐药 HIV 的质谱检测

• 批准号: 6742214
• 负责人: SADANAND GITE
• 金额: $ 29.99万
• 依托单位: AMBERGEN, INC
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-01 至 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6742214
• 关键词: RNA directed DNA polymerase; diagnostic tests; drug resistance; endopeptidases; gene mutation; genetic transcription; human immunodeficiency virus 1; mass spectrometry; matrix assisted laser desorption ionization; protein sequence; technology /technique development; virus protein

DESCRIPTION (provided by applicant): There are approximately one million persons in the U.S. infected with HIV-1, with 40,000 new cases reported each year. While most of these patients greatly benefit from highly active antiretroviral therapy (HAART), they often develop multi-drug resistance associated with viral mutations, which causes problems for long-term treatment. Knowledge of the spectrum of HIV-1 retroviral mutations is extremely important in order to effectively plan drug treatment for these patients. However sequencing is expensive, time-consuming and does not detect low-levels of drug-resistant strains. The principal objective of this proposal is to develop a highly sensitive, cost-effective and high throughput system for detecting drug-resistant strains of HIV-1 virus based on call-free protein expression and mass spectrometry. This approach is based on the fact that drug resistant inducing mutations in HIV alter the mass of the encoded proteins. During Phase I, the overall approach will be extensively evaluated using wild type(HXB2) and mutant sequences of the drug targeted viral reverse transcriptase and protease. Peptides from regions of each protein likely to contain drug-resistant mutations will be expressed in cell-free extracts from rabbit reticulocyte or E. coli using RT-PCR amplified DNA templates. In order to reduce proteolyUc degradation of cell-free expressed peptides, ia problem revealed in preliminary studies, a high fidelity reconstituted translation system based on unmodified E. coli translation factors will be tested. Several methods for rapid peptide fragment isolation from the cell-free extract will be evaluated based on earlier work by AmberGen including: i) N-terminal epitope incorporation, ii) tRNA mediated protein engineering (TRAMPE) and iii) photocleavable biotin. Mass spectrometric analysis of the purified HIV peptides will be performed using MALDI-TOF mass spectrometry both on individual peptides and multiple peptides produced by multiplexing PCR and all downstream steps. Results will be compared to other methods of HIV retrovirus genotyping including DNA sequencing. In Phase II, a high throughput MS based system will be developed for HIV-1 drug-resistance testing based on the industry standard 96- or 384-well sample format. Dr. Richard D'Aquila, an internationally recognized expert on HIV-1 drug-resistance will serve as a consultant on this project and as a source of samples. Dr. Raymond Tellier, an infectious disease expert who has used mass spectrometry to characterize quasi-species variation in Hepatitis C Virus will also serve as a consultant.

描述（由申请人提供）：美国大约有100万人感染了HIV-1，每年有40,000例新病例。尽管这些患者中的大多数大多数受益于高度活性的抗逆转录病毒疗法（HAART），但他们经常会产生与病毒突变相关的多药耐药性，这会导致长期治疗问题。为了有效地计划这些患者的药物治疗，HIV-1逆转录病毒突变谱的知识非常重要。但是，测序是昂贵的，耗时的，并且无法检测到低水平的抗药性菌株。 该提案的主要目标是开发一种高度敏感，成本效益和高吞吐量系统，用于基于无呼叫蛋白表达和质谱法检测HIV-1病毒的抗药性菌株。这种方法基于以下事实：抗艾滋病毒中抗药性突变改变了编码蛋白的质量。在第一阶段，将使用野生型（HXB2）和药物靶向病毒逆转录酶和蛋白酶的突变序列对整体方法进行广泛评估。使用RT-PCR扩增的DNA模板，来自每种蛋白质区域的肽将在兔网状细胞或大肠杆菌的无细胞提取物中表达。为了减少无细胞表达肽的蛋白水解降解，在初步研究中揭示了IA问题，将测试基于未修改的大肠杆菌翻译因子的高忠诚度重构翻译系统。将根据Ambergen的早期工作评估从无细胞提取物中分离出快速肽片段的几种方法，包括：i）N末端表位掺入，ii）tRNA介导的蛋白质工程（Trampe）和III）可透明生物素。纯化的HIV肽的质谱分析将使用MALDI-TOF质谱进行单个肽和多种PCR和所有下游步骤产生的多种肽进行。结果将与其他HIV逆转录病毒基因分型方法进行比较，包括DNA测序。在第二阶段，将根据行业标准96或384孔样品格式开发基于高吞吐量的MS系统。国际公认的HIV-1药物抗药性专家Richard D'Aquila博士将作为该项目的顾问和样本来源。传染病专家雷蒙德·泰勒（Raymond Tellier）博士使用质谱法来表征丙型肝炎病毒的准物种变化，也将作为顾问。