An establishment of cell line with high sensitivity to rotavirus by transfection of receptor gene and the application for diagnosis in rotavirus infection

受体基因转染建立轮状病毒高敏感性细胞系及其在轮状病毒感染诊断中的应用

• 批准号: 07556121
• 负责人: MINAMOTO Nobuyuki
• 金额: $ 1.98万
• 依托单位: Gifu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07556121/
• 关键词: Rotavirus; Sialic acid; Mucin; Molecular cloning; Anti-receptor antibody; Expression of gene; VP4; Ganglioside; 欠損VP4; 牛乳ムチン; MA-104細胞; 発現; レセプター遺伝子; クローニング; モノクローナル抗体; 腸管上皮細胞

In this study, to establish cell lines with high sensitivity to rotavirus, we determined complete sequence of all genomic segments of avian rotavirus, and characterization of a receptor for avian rotavirus on MA-104 cells. Moreover, we attempted to produce monoclonal antibodies directed to receptor for rotavirus. The ressults run as follows :(1) The complete nucleotide sequence of 11 genomic segments of avian rotavirus, P0-13 strain, which was isolated from a pigeon in our laboratory, was determined to elicit binding protein of rotavirus to cellular receptor. Then, the cloning genes encoding several proteins of rotavirus were expressed at a high level in simian COS7 cells and the expressed proteins were examined in the function of receptor binding.(Arch.Virol., 1995. Virology, 1995. Vet.Microbiol., 1996. Arch.Virol., 1996. Virus Genes, 1997. Virus Res., 1997)(2) By the treatment of MA-104 cell monolayrs with several enzymes, and by binding activity of rotaviruses to gangliosides, we suggested that sialic acid served as the surface molecule used by the virus for binding and entry into host cells.(Arch.Virol., in preparation)(3) It was indicated that ovomucin in hen egg white and mucin in cow milk bind to avian rotavirus, and these mucins inhibit infection of the virus to MA-104 cells. Although we produced many of monoclonal antibodies to these mucins, all of the resulting antibodies failed to inhibit infection of rotavirus to MA-104 cells so far.In the future, we will make an attempt to map receptor binding region in VP8 of rotavirus and to produce monoclonal antibodies against the synthetic peptides corresponding to the amino acid region

在本研究中，为了建立对轮状病毒高度敏感的细胞系，我们确定了禽类轮状病毒所有基因组片段的完整序列，并表征了 MA-104 细胞上禽类轮状病毒的受体。此外，我们尝试生产针对轮状病毒受体的单克隆抗体。结果如下：(1)测定了本实验室从鸽子中分离得到的禽轮状病毒P0-13株11个基因组片段的完整核苷酸序列，确定了轮状病毒与细胞受体的结合蛋白。然后，编码轮状病毒几种蛋白的克隆基因在猿猴COS7细胞中高水平表达，并检查表达的蛋白与受体结合的功能。(Arch.Virol., 1995. Virology, 1995. Vet.Microbiol., 1996. Arch.Virol.，1996.病毒基因，1997.病毒研究， 1997)(2)通过用几种酶处理MA-104单层细胞，并通过轮状病毒与神经节苷脂的结合活性，我们建议唾液酸作为病毒用于结合和进入宿主细胞的表面分子。（ Arch.Virol.，准备中)(3)表明鸡蛋清中的卵粘蛋白和牛奶中的粘蛋白与禽轮状病毒结合，这些粘蛋白抑制感染将病毒转移到 MA-104 细胞中。尽管我们针对这些粘蛋白生产了许多单克隆抗体，但迄今为止所有所得抗体均未能抑制轮状病毒对MA-104细胞的感染。将来，我们将尝试定位轮状病毒VP8中的受体结合区域，并产生针对对应氨基酸区域的合成肽的单克隆抗体

项目成果

Ito, H.: "Sequence analysis of the VP6 gene in group A turkey and chicken rotaviruses" Virus Research. 47. 79-83 (1997)

Ito, H.：“A 组火鸡和鸡轮状病毒中 VP6 基因的序列分析”病毒研究。

Ito H.,: "Mapping of antigenic sites on the major capsid protein of avian rotavirus using Escherichia coli expression system." Archives of Virology. (発表予定).

Ito H.，：“使用大肠杆菌表达系统绘制禽类轮状病毒主要衣壳蛋白的抗原位点。”（即将出版）。

Ito,H.: "Sequence analysis of cDNA for the VP6 protein of group A avian rotavirus:a comparison with group A mammalian rotaviruses." Archives of Virology. 140. 605-612 (1995)

Ito,H.：“A 组禽类轮状病毒 VP6 蛋白的 cDNA 序列分析：与 A 组哺乳动物轮状病毒的比较。”

Song,X,G.: "Antiviral activities in bovine colostrum that prevent human rotavirus infection in vivo." Milchwissenschaft. (発表予定).

Song, X, G.：“牛初乳中的抗病毒活性可预防人体内轮状病毒感染。”（待提交）。

Rohwedder A.,: "VP7 from rotavirus 993/83 isolated from a calf with diarrhea is closely related to VP7 of pigeon rotavirus PO-13." Virology. (発表予定).

Rohwedder A.，：“从腹泻小牛中分离出的轮状病毒 993/83 的 VP7 与鸽子轮状病毒 PO-13 的 VP7 密切相关。”