VACCINIA VIRUS DNA RECOMBINATION AND RECOMBINANTS

痘苗病毒 DNA 重组和重组

• 批准号: 2060663
• 负责人: L A BALL
• 金额: $ 28.4万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-08-01 至 1997-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2060663
• 关键词: DNA replication origin; Escherichia coli; RNA biosynthesis; cell free system; enzyme mechanism; gene expression; gene mutation; genetic manipulation; genetic mapping; genetic promoter element; genetic recombination; genetic regulation; genetic transcription; genetic translation; messenger RNA; mutant; nucleic acid sequence; replicase; thymidine kinase; tissue /cell culture; vaccinia virus; virus DNA; virus genetics; virus replication

The experiments described in this proposal are designed (1) to elucidate the mechanism of homologous DNA recombination in the orthopoxvirus vaccinia virus, and (2) to construct a novel type of recombinant vaccinia virus vector that provides " runaway" expression of foreign genes. As well as increasing our understanding of the mechanism of a fundamental genetic process. these experiments will permit the design of safer and more effective vaccines based on recombinant vaccinia viruses. Vaccinia virus represents one of the very few systems in which DNA recombination in higher eukeryotic cells is accessible to both a genetic and a biochemical approach. We therefore plan to isolate recombination-deficient (rec-) mutants of the virus and to examine the structures and functions of the rec gene products using both infected cells in culture and cell-free systems. Deletion of viral rec genes that are not essential for virus replication will enhance the safety of potential vaccines that are based on vaccinia virus recombinants. In the second part of the proposal, plans are described for the construction of a vaccinia virus recombinant that expresses the gene for an RNA-dependent RNA polymerase from a nodavirus. This enzyme normally catalyzes the replication of its own mRNA and should thus be expressed at very high levels from vaccinia virus recombinants. The possibility will be explored that RNA sequences that encode proteins of general interest and importance can be rendered competent templates for replication by the nodavirus replicase by being sandwiched between sequences that function as origins of nodavirus RNA replication. It is hoped that this approach will head to the development of new high-level expression vectors and potential vaccines based on vaccinia virus recombinants.

本提案中描述的实验设计为（1） 阐明同源DNA重组的机制， 正痘病毒牛痘病毒，和（2）构建新类型 重组牛痘病毒载体， 外源基因的表达。 除了增加我们的 了解一个基本的遗传机制， 过程 这些实验将允许设计更安全和 基于重组牛痘病毒的更有效的疫苗。 牛痘病毒是极少数几种系统之一， 高等真核细胞中的DNA重组是可利用的， 遗传学和生物化学的方法。 因此，我们计划 分离病毒的重组缺陷型（rec-）突变体， 检测rec基因产物的结构和功能 使用培养中的感染细胞和无细胞系统。 删除对病毒非必需的病毒rec基因 复制将提高潜在疫苗的安全性， 基于牛痘病毒重组体。 在提案的第二部分，介绍了 构建表达本发明的牛痘病毒重组体， 来自野田病毒的RNA依赖性RNA聚合酶的基因。 这种酶通常催化自身mRNA的复制 因此应该从牛痘中以非常高的水平表达 病毒重组体 将探索RNA 编码普遍感兴趣和重要的蛋白质的序列 可以提供合格的模板，供 野田病毒复制酶被夹在序列之间， 作为诺达病毒RNA复制的起点。 人们希望 这种方法将有助于开发新的高级别 基于牛痘病毒的表达载体和潜在疫苗 重组体