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Development of the system for the formation of infectious particles of rotavirus with the aid of genetic engineering

借助基因工程开发轮状病毒感染性颗粒形成系统

基本信息

批准号：
14370105
负责人：
TANIGUCHI Koki
金额：
$ 8.77万
依托单位：
FUJITA HEALTH UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

At the final stage of the research project, we succeeded in the development of the system of reverse genetics of rotavirus, although we have to employ a helper virus for the system. The methodology for the development is the following. 1) We prepared a plasmid (termed as pT7/VP4-SA11-L2) which the VP4 gene from a trypsin-independent simian rotavirus SA11-L2 clone with the ability of efficient multiplicity in cultured cell was inserted. 2) Vaccinia virus expressing T7 RNA polymerase was infected into COST cells. 3) A plasmid pT7/VP4(SA11-L2) was transfected. 4) A human rotavirus strain KU as a helper virus was infected into COST cells, and the cells were cultured for two days. 5) The culture fluid from the virus-infected cells was infected to MA-104 cells, and the cells were maintained for two days in the presence of a neutralizing monoclonal antibody YO-2C2, which inhibits specifically the growth of a helper virus KU. 6) RNA pattern of the RNA extracted from the culture fluid was confirmed by polyacrylamide gel electrophoresis.By these procedures, we succeeded in the preparation of infectious rotavirus particles which the VP4 gene was artificially inserted. Next, we prepared infectious rotaviruses with artificially mutated VP4 gene. The mutated VP4 genes are the one (pT7/VP4(SA11-L2)-Δ PstI) which a PstI restriction site on the gene was removed, and the other (pT7/VP4(SA11-L2)-Δ HaeII) whose a HaeII restriction site was removed by mutagenesis.Thus, we first developed the reverse genetics system for rotavirus. We try to prepare the infectious rotavirus with the mutated VP7 gene or NSP1 gene, and to prepare the infectious rotavirus with the aid of only plasmid DNAs in near future.

在研究项目的最后阶段，我们成功地开发了轮状病毒的反向遗传学系统，尽管我们必须为该系统使用辅助病毒。开发的方法如下。1)将胰酶非依赖性猴轮状病毒SA11-L2克隆中的VP4基因插入pT7/VP4-SA11-L2中，构建成重组表达载体pT7/VP4-SA11-L2。2)将表达T7 RNA聚合酶的痘苗病毒感染COST细胞。3)构建表达载体pT7/VP4(SA11-L2)。4)将人轮状病毒KU株作为辅助病毒感染COST细胞，培养2天。5)将病毒感染细胞的培养液感染MA-104细胞，在特异性抑制辅助病毒KU生长的中和性单抗YO-2C2存在下培养两天。6)用聚丙烯酰胺凝胶电泳法对从培养液中提取的RNA进行了鉴定，成功地制备了人工插入VP4基因的感染性轮状病毒颗粒。接下来，我们用人工突变的VP4基因制备了传染性轮状病毒。突变的VP4基因分别是Pt7/VP4(Sa11-L2)-ΔPstI基因和Pt7/VP4(Sa11-L2)-ΔHaeII基因。我们尝试用突变的VP7基因或NSP1基因制备传染性轮状病毒，并在不久的将来仅用质粒DNA制备传染性轮状病毒。

项目成果

期刊论文数量（64）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Adah MI: "Close relationship between G8 bovine and human rotaviruses isolated in Nigeria"Journal of Clinical Microbiology. 41・8. 3945-3950 (2003)

Adah MI：“尼日利亚分离的 G8 牛和人轮状病毒之间的密切关系”临床微生物学杂志 41・8（2003）。

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0
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J.Sasaki, K.Taniuuchi: "The 5'-end sequence of the genome of Aichi virus, a Picornavirus, contains an element Critical for viral RNA encapsidation"Journal of Virology. 77・6. 3542-3548 (2003)

J.Sasaki、K.Taniuuchi：“爱知病毒（一种小核糖核酸病毒）基因组的 5 端序列包含对病毒 RNA 衣壳化至关重要的元件”《病毒学杂志》77·6 (2003)。

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0
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Protective efficacy of a sulfated sialyl lipid (NMSO3) against human rotavirus-Induced diarrhea in a mouse model.

硫酸化唾液酸脂质 (NMSO3) 对小鼠模型中人轮状病毒引起的腹泻的保护作用。

DOI：
发表时间：
2002
期刊：
Antimicrob Agents Chemother 46(2)
影响因子：
0
作者：
Takahashi K;Ohashi K;Abe Y;Mori S;Taniguchi K;Ebina T;Nakagomi 0;Terada M;Shigeta S
通讯作者：
Shigeta S

Y.Pongsuwanna et al.: "Detection of a human rotavirus with G12 and P[9] specificity in Thailand"Journal of Clinical Microbiology. 40・4. 1390-1394 (2003)

Y. Pongsuwanna 等人：“在泰国检测具有 G12 和 P[9] 特异性的人类轮状病毒”《临床微生物学杂志》40・4（2003 年）。