Application and development of molecular biological meth

分子生物学方法的应用与发展

• 批准号: 6678857
• 负责人: KEITH 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/6678857
• 关键词: RNA directed DNA polymerase; Retroviridae; bioassay; biological products; chemical structure function; coagulation factor VIII; drug quality /standard; drug screening /evaluation; enzyme activity; host organism interaction; human immunodeficiency virus 1; immunologic substance development /preparation; molecular biology; nucleic acid quantitation /detection; polymerase chain reaction; public health; technology /technique development; time resolved data; tissue /cell culture; virus infection mechanism; virus protein

Summary: The goal of this project is to apply and develop molecular biological methods and animal models that can ensure that vaccines and cell substrates are free from viral, and particularly retroviral, contamination and to assess the oncogenic and infectivity risks associated with residual cell-substrate DNA. Reverse transcriptase (RT) assays measure the presence of retroviruses. The conventional RT assay was insenstive and not quantitative. This changed with the advent of PCR-based RT PBRT) assays, which are a million-fold more sensitive than conventional RT assays. We set up the three PBRT assays at CBER in order to compare their sensitivities, specificities, and reproducibilities. Modifications were made to the assays to eliminate one problem with the assays, viz., their high background signals. We also modified the assay such that RT activities of cellular DNA polymerases were substrantially reduced. 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 assay, the TM-PERT assay, is linear over at least 6 orders magnitude and is as sensitive as the original assays. As part of our investigation into the chick RT activity, we began a study to determine whether pseudotypes can form between a retrovirus core and an envelope glycoprotein (Env) from paramyxoviruses or orthomyxoviruses. If so, then this may provide a means by which the chick RT particle could enter human cells. As a model system, we investigated measles, mumps and influenza virus Envs with HIV core particles. Our results show that pseudotypes can form in vitro, and thus there is a theoretical possibility of such pseudotypes forming in vivo. The consequences of such pseudotype formation remains unknown. We have demonstrated that mixed pseudotypes [i.e., pseudotypes containing envelope glycoproteins (Envs) from two different viruses] can form between measles virus and HIV-1 Envs. This raises the possibility that such virions can form in vivo. This may have implications for HIV pathogenesis, since HIV particles containing an Env of measles virus would be able to infect non-CD4 cells; such infected cells would have unknown consequences on HIV pathogenesis. We have developed Q-PCR assays to detect the genomes of primate polyomaviruses (SV40, JCV, BKV). Assays for these viruses will be a first step in our program to establish quantitative assays for the detection of adventitious agents. With these Q-PCR assays, we are quantifying the levels of the human polyomaviruses BKV and JCV DNA in human blood with the aim of determining whether the primate polyomavirus SV40 is present in human blood. We have shown that antibodies to SV40 can neutralise BKV and antibodies to BKV can neutralise SV40. We are developing a reporter-gene neutralisation assay for SV40, BKV, and JCV in order to test whether exposure to one polyomavirus can affect the probability of being infected with another polyomavirus. We have initiated studies to develop quantitative assays to assess the biological activity of residual cell-substrate DNA. For many years, DNA resulting from cell substrates has been considered to pose a risk to vaccine recipients receiving products manufactured in neoplastic cells. This is one of the main reasons for these cells not being used for vaccine production to date. The risk is perceived to be either from an oncogenic potential and an infectious potential. We are developing quantitaive assays to assess both types of risks. In collaboration with NCI and CDER, we are exploring animal models to asssess DNA oncogenicity. We have developed an in vitro system to quantify the infectivity of retroviral DNA.

总结：该项目的目标是应用和开发分子生物学方法和动物模型，以确保疫苗和细胞基质不受病毒（特别是逆转录病毒）污染，并评估与残留细胞基质DNA相关的致癌性和感染性风险。 逆转录酶（RT）测定可测量逆转录病毒的存在。 传统的RT分析是不敏感的，不能定量。 这随着基于PCR的RT（PBRT）测定的出现而改变，其比常规RT测定灵敏一百万倍。 我们在CBER建立了三种PBRT检测方法，以比较它们的灵敏度、特异性和重现性。 对测定法进行了修改以消除测定法的一个问题，即，高背景信号。 我们还修改了测定法，使RT细胞DNA聚合酶的活动大幅减少。 最近，我们已将PBRT测定法与真实的时间定量系统TaqMan系统一起使用，以与Perkin-Elmer 7700系统一起使用。 这种改进的检测方法（TM-PERT检测方法）在至少6个数量级上呈线性，并且与原始检测方法一样灵敏。 作为我们对鸡RT活性的调查的一部分，我们开始了一项研究，以确定是否可以在逆转录病毒核心和副粘病毒或正粘病毒的包膜糖蛋白（Env）之间形成假型。 如果是这样的话，那么这可能提供了一种方法，通过这种方法，鸡RT颗粒可以进入人类细胞。 作为一个模型系统，我们研究了麻疹，腮腺炎和流感病毒与HIV核心颗粒的Envs。 我们的研究结果表明，假型可以在体外形成，因此有一个理论上的可能性，这样的假型在体内形成。 这种假型形成的后果仍然未知。 我们已经证明了混合伪型[即，包含来自两种不同病毒的包膜糖蛋白（Envs）的假型]可以在麻疹病毒和HIV-1 Envs之间形成。 这增加了这种病毒体可以在体内形成的可能性。 这可能对HIV发病机制有影响，因为含有麻疹病毒Env的HIV颗粒能够感染非CD 4细胞;这种感染的细胞对HIV发病机制有未知的后果。 我们已经开发了Q-PCR检测灵长类多瘤病毒（SV 40，JCV，BKV）的基因组。 这些病毒的检测将是我们建立外源因子定量检测方法的第一步。 通过这些Q-PCR检测，我们正在定量人血液中人多瘤病毒BKV和JCV DNA的水平，目的是确定人血液中是否存在灵长类多瘤病毒SV 40。 我们已经证明，抗SV 40的抗体可以中和BKV，抗BKV的抗体可以中和SV 40。 我们正在开发SV 40、BKV和JCV的病毒基因中和试验，以测试暴露于一种多瘤病毒是否会影响感染另一种多瘤病毒的可能性。 我们已经开始研究开发定量分析，以评估残留的细胞底物DNA的生物活性。 多年来，细胞基质产生的DNA被认为对接受肿瘤细胞生产产品的疫苗接种者构成风险。 这是迄今为止这些细胞未用于疫苗生产的主要原因之一。 风险被认为是来自致癌潜力和感染潜力。 我们正在开发定量分析来评估这两种类型的风险。 在与NCI和CDER的合作中，我们正在探索评估DNA致癌性的动物模型。 我们已经开发了一种体外系统来量化逆转录病毒DNA的感染性。