DEVELOPMENT OF MOLECULAR BIOLOGICAL METHODS TO VACCINE AND CELL SUBSTRATE SAFETY

疫苗和细胞基质安全的分子生物学方法的开发

• 批准号: 6101188
• 负责人: K PEDEN
• 金额: --
• 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/6101188
• 关键词: Retroviridae; drug quality /standard; human tissue; nucleic acid sequence; polymerase chain reaction; vaccines

The goal of this project is to apply and develop molecular biological methods that can ensure that vaccines and cell substrates are free from viral, and particularly retroviral, contamination. The recent publications of three sensitive methods for the detection of reverse transcriptase (RT) that are at least a million-fold more sensitive than conventional RT assays and the finding using one of these assays that certain vaccines had detectable levels of RT activity necessitated that CBER undertake research to address this issue. The three assays depend on the same principle: an RNA of known sequence is used as a template with an oligodeoxynucleotide primer in an RT reaction; because the sequence of the RNA is known, the cDNA product can be amplified by the polymerase chain reaction (PCR); the PCR product can be detected by a number of methods. The three assays use different RNA templates: brome mosaic virus (BMV) genome, MS2 genome, and an in vitro synthesized product from encephalomyocarditis virus (EMCV). Although the assays were variously termed PERT (for product-enhanced RT) or Amp-RT, we have chosen to call them by the generic name of PBRT for PCR-based RT. We set up the three assays at CBER in order to compare their sensitivities, specificities, and reproducibilities. We found that the MS2 assay (PERT) was about 10 to 50 times more sensitive than the BMV assay, which was about 10 to 100 fold more sensitive than the Amp-RT assay. By increasing the template-primer levels of the BMV assay to those of the PERT assay, the two assays became equally sensitive. In addition, we designed primers to be used with a third commercially-available RNA, TMV, and the assay with this template was similarly sensitive. The inherent background signal of the assay was effectively eliminated by incorporating an RNase digestion prior to the PCR step and using a thermostable polymerase lacking RT activity and. Because of the high sensitivity of the PBRT assay, cell extracts produce a positive signal as do many DNA-dependent DNA polymerases. Reasoning that genuine RT~s would have properties that would enable the enzyme to copy RNA, we designed primers that were around regions of secondary structure and that required that a longer cDNA had to be made. With this modified assay, the signal produced by the cell extract was diminished several logs without significant loss in sensitivity. Importantly, retroviruses from all known classes scored positive. Recently, we have adapted the PBRT assay for use with the real time quantitative system, the TaqMan system, for use with the Perkin-Elmer 7700 system. This modified PBRT assay, the TM-PERT assay, is linear over at least 6 orders magnitude and is as sensitive as the original PBRT assays. We are currently trying to modify this assay to make it selective for retroviruses.

本项目的目标是应用和发展分子生物学 可以确保疫苗和细胞基质不含 病毒，特别是逆转录病毒的污染。 本文综述了近年来发表的三种灵敏的检测大肠杆菌的方法， 逆转录酶（RT），至少有一百万倍以上 比传统的RT检测更敏感，使用其中之一的发现 某些疫苗具有可检测水平的RT活性 需要CBER进行研究来解决这个问题。 的 三种测定法依赖于相同的原理：已知序列的RNA， 在RT反应中与寡脱氧核苷酸引物一起作为模板; 因为RNA的序列是已知的，所以cDNA产物可以是 通过聚合酶链式反应（PCR）扩增; PCR产物可以是 通过多种方法检测。 这三种检测方法使用不同的RNA 模板：雀麦花叶病毒（BMV）基因组、MS 2基因组和体外 脑心肌炎病毒（EMCV）的合成产物。 虽然 这些测定被不同地称为PERT（用于产物增强RT）或 Amp-RT，我们选择用PBRT的通用名称来称呼它们， 基于PCR的RT 我们在CBER建立了三种检测方法，以比较它们的 敏感性、特异性和重现性。 我们发现 MS 2试验（PERT）的灵敏度比BMV高约10至50倍 检测，这是约10至100倍更敏感的Amp-RT 比色法 通过增加BMV测定的模板-引物水平， 与PERT测定的那些相比，这两种测定变得同样灵敏。 在 此外，我们设计了引物，用于第三个 商业上可获得的RNA，TMV，并且使用该模板的测定是 同样敏感。 测定的固有背景信号为 通过在将RNA酶消化之前引入RNA酶消化而有效地消除， PCR步骤，并使用缺乏RT活性的热稳定聚合酶，以及。 由于PBRT测定的高灵敏度，细胞提取物产生 许多依赖DNA的DNA聚合酶也是阳性信号。 推理 真正的逆转录酶应该具有这样的特性， 复制RNA，我们设计了引物， 结构，这需要一个更长的cDNA。 与此 在改良的测定中，细胞提取物产生的信号减弱 几个日志没有显着损失的灵敏度。 重要的是， 所有已知种类的逆转录病毒均为阳性。 最近我们 使PBRT测定适合与真实的时间定量系统一起使用， TaqMan系统，用于Perkin-Elmer 7700系统。 这 改良的PBRT测定，即TM-PERT测定，在至少6个数量级上呈线性 该方法的灵敏度与原始PBRT测定法相同。 我们 目前正在尝试修改这种检测方法，使其对 逆转录病毒