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STUDIES ON THE STRUCTURE AND FUNCTION OF NONSTRUCTURAL PROTEINS OF ROTAVIRUS

轮状病毒非结构蛋白的结构和功能研究

基本信息

批准号：
07457080
负责人：
TANIGUCHI Koki
金额：
$ 4.93万
依托单位：
SAPPORO MEDICAL UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

项目摘要

1.We isolaed subclones producing large, medium, or small plaques from each of A5-13 expressing normal NSP1, A5-10 encoding omly 40 amino acid-NSP1, and A5-16 having NSP1 gene with 500 bp deletion. All of the clones replicated well in MA-104 cells, and induced dirrhea in suckling mice by oral administration. These results indicate that the insertion of foreign gene into NSP1 gene will be possible when the system of artificial infectious virus particles is established.2.We first determined the complete nucleotide sequence of the genome of human ratavirus (strain KU).3.All of the 6 structural proteins VP1-VP4, VP6, VP7 and 4 of 5 non-structural proteins of human rotavirus were expressed in baculovirus expression system. The produre for purification of his-tagged nonstructural proteins was established, and this enabled the precise examination of the function of nonstructural proteins.4.Self-assembled, artificial and empty single-shelled particles or double-shelled particules were obtained by coexpressions of VP2 and VP6, or VP2, VP6 and VP7. In addition, the coexpression of VP1, VP3, VP6, VP7, and VP4 as well as VP2 resulted in the formation of artificial virus-like particles lacking genome. These artificial empty particles have the value as a value as a vaccine candidate and a tool for studying the immunity of rotavirus infection.5.In the infection experiments by oral inoculation to suckling mice, we observed the diarrhea in the mice administered with G1, G3, G4, G8, and G12 human rotavirus strains. This finding show that it is possible to carry out the rotavirus infection-protection experiments in mice by using human rotaviruses.

1.我们从表达正常NSP1的A5-13、编码全部40个氨基酸-NSP1的A5-10和具有500bp缺失的NSP1基因的A5-16中分离出产生大、中或小噬斑的亚克隆。所有克隆在 MA-104 细胞中复制良好，并通过口服诱导乳鼠腹泻。这些结果表明，当人工感染病毒颗粒系统建立后，将外源基因插入NSP1基因将成为可能。2.我们首次测定了人轮状病毒（KU株）基因组的完整核苷酸序列。3.人轮状病毒的6个结构蛋白VP1-VP4、VP6、VP7以及5个非结构蛋白中的4个均在杆状病毒中表达。表达系统。建立了his标记的非结构蛋白纯化工艺，为非结构蛋白功能的精确检测提供了可能。4.通过VP2和VP6，或VP2、VP6和VP7共表达，获得自组装的人工空心单壳颗粒或双壳颗粒。此外，VP1、VP3、VP6、VP7、VP4以及VP2的共表达导致形成缺乏基因组的人工病毒样颗粒。这些人工空颗粒具有作为候选疫苗的价值和研究轮状病毒感染免疫的工具的价值。 5.在乳鼠口服接种感染实验中，我们观察到给予G1、G3、G4、G8和G12人轮状病毒株的小鼠出现腹泻。这一发现表明，利用人轮状病毒在小鼠体内进行轮状病毒感染保护实验是可能的。