STUDIES ON THE STRUCTURE AND FUNCTION OF NONSTRUCTURAL PROTEINS OF ROTAVIRUS

1.从表达正常NSP 1的A5-13、编码仅40个氨基酸的NSP 1的A5-10和缺失500 bp的NSP 1基因的A5-16中分离出产生大、中、小噬斑的亚克隆。所有克隆在MA-104细胞中均能良好增殖，并经口给药诱导乳鼠腹泻。2.首次测定了人轮状病毒KU株全基因组的核苷酸序列; 3.在杆状病毒表达系统中表达了人轮状病毒VP 1-VP 4、VP 6、VP 7共6个结构蛋白和5个非结构蛋白中的4个。4.通过共表达VP 2和VP 6，或VP 2、VP 6和VP 7，获得了自组装的、人工的、空的单壳颗粒或双壳颗粒。此外，VP 1、VP 3、VP 6、VP 7和VP 4以及VP 2的共表达导致了缺乏基因组的人工病毒样颗粒的形成。这些人工空颗粒具有作为轮状病毒疫苗候选物和研究轮状病毒感染免疫的工具的价值。5.在口服接种乳鼠的感染实验中，我们观察到G1、G3、G4、G8和G12人源轮状病毒株感染的小鼠出现腹泻。这一发现表明，有可能通过使用人轮状病毒在小鼠中进行轮状病毒感染保护实验。

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.