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Molecular Biologic and Molecular Epidemiologic Study of Group A Human Rotavirus

A组人轮状病毒的分子生物学和分子流行病学研究

基本信息

批准号：
62570205
负责人：
URASAWA Shozo
金额：
$ 1.34万
依托单位：
Sapporo Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-62570205/
关键词：
Human rotavirus Monoclonal antibody Neutralization epitope Serotype antigen Cross reactive antigen Viral protein Viral RNA 塩基配列

项目摘要

1. By immunizing human rotavirus(HRV), we established 62 hybridomas that secrete monoclonal antibodies (Mabs) neutralizing HRV. Based on the reactivity patterns of these Mabs against various HRV strains in neutralization and enzyme-linked immunosorbent assays, they were classified into either serotype-specific or cross-reactive Mabs. Protein specificity of these mabs determined by both immunoprecipitation analysis and the use of genetically reassorted HRV strains revealed that while the majority of serotype-specific antigens resided for the most part in VP7 and partly (only a part of serotype 2-specific antigen) in VP3, cross-reactive antigens were located mostly in VP3 and partly in VP7. 2. By using these Mabs we selected antigenic mutants of KU strain (serotype 1 HRV) that resisted neutralization by the Mabs used for their selection. Cross-neutralization tests between the VP7-derected mabs and the antibody-selected antigenic mutants carried out to date identified one cross-reactive a … More nd five distinct serotype-specific neutralization epitopes which operationally overlapped one another and constituted a single antigenic site in VP7. The amino acid substitution in VP7 that are responsible for the antigenic alterations in the mutants were identified from sequencing of VP7 genes of the wild and mutant KU strains. All the amino acid substitutions in the antigenic mutants occurred in one of the two variable regions: amino acid 87 to 101 and 208 to 221.Cross-neutralization tests between the VP3-directed Mabs and the corresponding antigenic mutants carried out to date identified at least three distinct cross-reactive neutralization epitopes in VP3. Sequencing of VP3 genes of the antigenic mutants also indicated three distinct neutralization epitopes in VP3: the mutants sustained a single amino acid substitution at position 305, 433 or 392 (or 439 which seemed to be located in close proximity to 392 in three dimensional structure). A synthetic peptide (amino acids 296 to 313) which included the sequence of the first neutralization epitope reacted with its corresponding Mab, suggesting that the region contains a "sequential" antigenic determinant. Less

1. 通过免疫人轮状病毒（HRV），我们建立了62株能分泌中和HRV的单克隆抗体（mab）的杂交瘤。根据这些单抗在中和和酶联免疫吸附试验中对各种HRV菌株的反应性模式，它们被分为血清型特异性单抗和交叉反应单抗。通过免疫沉淀分析和基因重组HRV菌株测定这些单抗的蛋白特异性，发现大多数血清型特异性抗原大部分位于VP7，部分位于VP3（只有一部分是2型特异性抗原），而交叉反应性抗原大部分位于VP3，部分位于VP7。2. 通过使用这些单抗，我们选择了KU菌株（血清型1 HRV）的抗原突变体，这些突变体可以抵抗用于选择的单抗的中和作用。在VP7检测的单抗和抗体选择的抗原突变体之间进行的交叉中和试验中，迄今为止鉴定出了一个交叉反应性的抗原表位和五个不同的血清型特异性中和表位，这些表位在操作上相互重叠，构成了VP7的单个抗原位点。通过对野生菌株和突变菌株VP7基因的测序，确定了VP7中导致突变株抗原性改变的氨基酸取代。所有抗原突变体的氨基酸替换都发生在两个可变区域中的一个：氨基酸87 ~ 101和208 ~ 221。迄今为止，VP3定向单克隆抗体和相应的抗原突变体之间的交叉中和试验确定了VP3中至少三个不同的交叉反应性中和表位。抗原突变体的VP3基因测序也显示了VP3中三个不同的中和表位：突变体在305、433和392位（或439在三维结构上似乎位于392附近）维持单个氨基酸替代。包含第一个中和表位序列的合成肽（氨基酸296至313）与其对应的单抗反应，表明该区域包含“顺序”抗原决定因子。少