PRODUCTION OF ATTENUATED VACCINE CANDIDATE DENGUE VIRUSES FROM INFECTIOUS CDNA

从传染性 CDNA 生产登革热病毒候选减毒疫苗

• 批准号: 3770341
• 负责人: B FALGOUT
• 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/3770341
• 关键词: attenuated microorganism; complementary DNA; dengue virus; gene mutation; genome; molecular cloning; nucleic acid chemical synthesis; nucleic acid sequence; oligonucleotides; polymerase chain reaction; viral vaccines; virus RNA; virus genetics

The dengue virus genome is a positive-strand RNA molecule of approximately 11 kb. Purified genomic RNA is infectious upon transfection into appropriate cells. The goal of this project is to clone a cDNA copy of the genome of dengue virus type 2, New Guinea C strain (DEN2 NGC) such that a faithful, infectious copy of the genome can be made by in vitro transcription using the commercially available SP6 RNA polymerase. Creatio of an infectious clone will allow introduction of defined mutations into th dengue genome. Such a capability would be useful in many types of studies, including mapping mutations responsible for altered host range in neurotropic strains of DEN2, and attempting to attenuate DEN2 for possible use as a vaccine. Stocks of DEN2 NGC have been grown and genomic RNA prepared. Primers for reverse transcription/polymerase chain reaction (RT PCR) have been designed and synthesized. We have tried to amplify relatively large segments of the genome, so that only a few cloned pieces have to be assembled to make a full-length copy. The best PCR results have been obtained using unprimed R reactions. The entire genome except for the right-hand 120 nucleotides has been successfully amplified. The left-hand 5.3 kb was amplified in one piece, but is not yet cloned. The right-hand 3 kb also has been amplified but not cloned. The next steps are to clone the remainder of the genome, chemically synthesize the right-hand 120 nucleotides, and then assemble a full-length clone from the pieces. Hopefully, in vitro transcription of this clone will produce infectious RNA.

登革热病毒基因组是一种正链RNA分子，大约 11kb。纯化的基因组RNA在转染腺病毒后具有传染性 适当的单元格。这个项目的目标是克隆一个cdna拷贝的 登革2型病毒新几内亚C株(DEN2NGC)基因组 在体外可以制造出忠实的、有感染力的基因组拷贝。 使用商业上可获得的SP6 RNA聚合酶转录。创作 感染性克隆将允许将已定义的突变引入 登革热基因组。这种能力在许多类型的研究中都是有用的， 包括导致宿主范围改变的图谱突变 DEN_2嗜神经性菌株，并试图减弱DEN_2 用作疫苗。 已经培养了DEN2NGC的库存，并制备了基因组RNA。适用于 设计了逆转录/聚合酶链式反应(RT-PCR) 并合成了。我们试图放大相对较大的片段 基因组，因此只需要组装几个克隆片段就可以形成 全长副本。利用未加引物的R-R获得了最佳的PCR结果 反应。除了右边的120个核苷酸外，整个基因组都有 已被成功扩增。扩增出左侧5.3kb的DNA片段 片段，但尚未克隆。右边的3kb也被扩增了 但不是克隆的。下一步是克隆剩余的基因组， 化学合成右侧的120个核苷酸，然后组装成一个 从碎片中进行全长克隆。希望，在体外转录的 这种克隆将产生具有感染性的RNA。