N-GLYCOSYLATION MECHANISM IN INSECT CELLS

昆虫细胞中的 N-糖基化机制

• 批准号: 2459493
• 负责人: Donald L. Jarvis
• 金额: $ 15.87万
• 依托单位: TEXAS A&M AGRILIFE RESEARCH
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1997-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2459493
• 关键词: Baculoviridae; Lepidoptera; SDS polyacrylamide gel electrophoresis; animal tissue; gene expression; glycoprotein biosynthesis; glycoprotein structure; glycosylation; insect virus; laboratory rabbit; mannosidase; oligosaccharides; recombinant proteins; tissue /cell culture; transfection /expression vector; virus diseases; western blottings

For the past decade, insect cells have been used to produce important recombinant glycoproteins for biomedical research. Meanwhile, insect molecular cell biology has received little attention. As a result, the glycoprotein processing capabilities of insect cells remain poorly defined, while their use for glycoprotein expression has become commonplace. This problem is exacerbated by recent reports which challenge our current views on glycoprotein processing in insect cells. The long-term objectives of this proposal are to provide a better understanding of the insect cell N-glycosylation pathway and to determine if and how baculovirus infection influences this pathway. The following specific aims will address these long-term objectives: 1. Determine the structures of N-linked oligosaccharides in a major baculovirus glycoprotein produced in: a. Insect cells. b. Mammalian cells. c. Insect cells modified to coexpress to mammalian processing enzyme. 2. Isolate and characterize insect cell alpha-mannosidase genes. 3. Characterize the enzymatic properties of insect cell alpha- mannosidases. 4. Assess the effect of baculovirus infection on expression of insect cell alpha-mannosidases. The health-relatedness of this proposal is significant due to the unlimited medical applications of the insect cell-baculovirus expression system. Glycoproteins produced in this system can be used as vaccines, diagnostic reagents, and/or therapeutic agents, and we must be able to accurately predict their structures. The proposed studies also will contribute new information to several different areas of basic insect science, including insect molecular cell biology, biochemistry, and virology. These findings will be generally applicable to medically important insects, like mosquitos, and could lead to the development of better ways to control them. Finally, this proposal will provide new information on the number, locations, and structures of the carbohydrate moieties in a major baculovirus glycoprotein. They also will begin to address the possible role of carbohydrates in the function of this important virion component, which is involved in the penetration of baculoviruses into insect cells and the systemic spread of virus infection within individual insect larvae.

在过去的十年中，昆虫细胞已被用来产生重要的 重组糖蛋白用于生物医学研究。 同时，昆虫 分子细胞生物学几乎没有受到关注。 结果， 昆虫细胞的糖蛋白加工能力仍然很差 定义，虽然它们用于糖蛋白表达的使用已成为 平凡。 最近的报告加剧了这个问题 挑战我们当前对昆虫细胞中糖蛋白加工的看法。 该提案的长期目标是提供更好的 了解昆虫细胞N-糖基化途径并确定 如果以及杆状病毒感染如何影响这一途径。 以下特定目标将解决以下长期目标： 1。确定主要的N连接寡糖的结构 杆状病毒糖蛋白在： 一个。 昆虫细胞。 b。 哺乳动物细胞。 c。 昆虫细胞被修饰以表达为哺乳动物加工酶。 2。分离和表征昆虫细胞α-甘露糖苷酶基因。 3。表征昆虫细胞α-的酶促特性 甘露酶。 4。评估杆状病毒感染对昆虫表达的影响 细胞α-甘露糖苷酶。 由于 昆虫细胞 - 巴克洛病毒表达的无限医学应用 系统。 该系统中产生的糖蛋白可以用作疫苗， 诊断试剂和/或治疗剂，我们必须能够 准确预测其结构。 拟议的研究也将 为基本昆虫的几个不同领域贡献新信息 科学，包括昆虫分子细胞生物学，生物化学和 病毒学。 这些发现通常适用于医疗 重要的昆虫，例如蚊子，可能导致 更好的方法来控制它们。 最后，该建议将提供新的 有关碳水化合物的数量，位置和结构的信息 主要杆状病毒糖蛋白中的部分。 他们也会开始 解决碳水化合物在此功能中的可能作用 重要的病毒成分，与渗透有关 杆状病毒进入昆虫细胞和病毒感染的全身传播 在单个昆虫幼虫中。