Study on the structure and function of viral proteins using artificial particles of human rotavirus

利用人轮状病毒人工颗粒研究病毒蛋白的结构和功能

• 批准号: 10470080
• 负责人: TANIGUCHI Koki
• 金额: $ 8.45万
• 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: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10470080/
• 关键词: ROTAVIRUS; BACULOVIRUS; PROTECTION; VACCINE; ARTIFICIAL PARTICLE; MUCOSAL ADJUVANT; CTL; REVERSE GENETICS; GFP遺伝子; オステオポンチン; 構造蛋白; RNAセグメント; 血清型

1. Self-assembled, artificial and empty single-shelled particles (VP2/6) were prepared by using baculovirus expression system, and they were purified in a large quantity. VP2/6 particles accompanied with mucosal adjuvant (mLT-135, mLT-H44A, recombinant B subunit) were intranasally immunized in mice. Significant IgG and IgA responses in serum and stool were observed when mLT-135 was used as a mucosal adjuvant. In the inoculation with viruent murine strain EW for the immunized mice, a marked decrease of virus antigen in stools was observed in the case of mLT-135. Furthermore, a strong CTL response was also observed. Expression of mRNA for Th1 or Th2 cytokines was detected in the spleen cells from the immunized mice. Thus, artificial empty single-shelled particles combined with mLT-135 induced efficiently both of humoral and cellular immune responses in mice, suggesting the utility of the artificial particles and mucosal adjuvant as a vaccine for humans against rotavirus infection.2. We o … More bserved that human rotaviruses with G serotype other than G3 also induced diarrhea in suckling mice. This finding is useful for infection experiments using human rotavirus strains in mice.3. We carried out the following experiments for trying to establish reverse genetics of rotavirus.(1) RNA transcripts from VP4 or VP7 gene of strain SAT11-L2 which is less dependent on trypsin were transfected to MA 104 cells with the aid of a helper virus, human strain KU which is highly dependent on trypsin.(2) We prepared several constructs for transfection : T7 promoter-rotavirus VP4 or VP7 gene-rybozyme from type D hepatitis virus-T7 terminater. Truncated NSP1 genes which are not necessarily required for replication were connected to picornavirus IRES and GFP gene, and they were inserted in the above plasmid. The RNA transcripts were transfected into MA 104 cells infected with human strain KU.The screening assay for detecting the cells expressing GFP or producing infectious reassortant virus was developed. However, infectious artificial rotavirus has not been successfuly isolated so far. Less

1.利用杆状病毒表达系统制备自组装的人工空单壳颗粒（VP2/6），并进行大量纯化。将VP2/6颗粒与粘膜佐剂（mLT-135、mLT-H44A、重组B亚基）一起鼻内免疫小鼠。当使用 mLT-135 作为粘膜佐剂时，在血清和粪便中观察到显着的 IgG 和 IgA 反应。在用强毒鼠株EW接种免疫小鼠时，在MLT-135的情况下观察到粪便中的病毒抗原显着减少。此外，还观察到强烈的 CTL 反应。在免疫小鼠的脾细胞中检测到 Th1 或 Th2 细胞因子 mRNA 的表达。因此，人工空单壳颗粒与MLT-135结合可有效诱导小鼠体液和细胞免疫反应，表明人工颗粒和粘膜佐剂可作为人类抗轮状病毒感染的疫苗。 2．我们观察到，除 G3 之外的 G 血清型人类轮状病毒也会引起乳鼠腹泻。这一发现对于在小鼠中使用人轮状病毒株进行感染实验很有用。3．我们进行了以下实验，试图建立轮状病毒的反向遗传学。(1)在辅助病毒、高度依赖胰蛋白酶的人类株KU的帮助下，将不太依赖胰蛋白酶的SAT11-L2毒株的VP4或VP7基因的RNA转录本转染到MA 104细胞中。(2)我们制备了几种用于转染的构建体：T7启动子-轮状病毒VP4或 来自D型肝炎病毒-T7终止子的VP7基因-核酶。将复制不一定需要的截短的NSP1基因与小核糖核酸病毒IRES和GFP基因连接，并将它们插入到上述质粒中。将RNA转录本转染至感染人KU株的MA 104细胞中。开发了检测表达GFP或产生感染性重配病毒的细胞的筛选方法。然而，至今尚未成功分离出具有传染性的人工轮状病毒。较少的

项目成果

N.Kobayashi: "Sequence analysis of structural and non structural proteins of a human group B rotavirus detected in Calcutta, India."J.Med.Virol.. (in press). (2001)

N.Kobayashi：“对印度加尔各答检测到的人类 B 组轮状病毒的结构和非结构蛋白进行序列分析。”J.Med.Virol..（出版中）。

J.Okada: "Preferential selection of heterologous G3-VP7 gene in the genetic background of simian rotavirus SA11 detected by using a homotypic single-VP7 gene-substitution reassortant."Antiviral Res.. 42. 712-720 (1998)

J.Okada：“通过使用同型单 VP7 基因取代重配检测到猿轮状病毒 SA11 遗传背景中异源 G3-VP7 基因的优先选择。”抗病毒研究 42. 712-720 (1998)

K.Takahashi: "Analysis of anti-rotavirus activity of extract from Stevia rebaudiana."Antiviral Res.. 49. 15-24 (2001)

K.Takahashi：“甜叶菊提取物的抗轮状病毒活性分析。”抗病毒研究 49. 15-24 (2001)

J.Okada et al.: "Analysis on reassortment of rotavirus NSP1 genes lacking coding region for cysteine-rich zinc finger motif."Arch.Virol.. 144. 345-353 (1999)

J.Okada 等人：“缺乏富含半胱氨酸的锌指基序编码区的轮状病毒 NSP1 基因重配分析。”Arch.Virol.. 144. 345-353 (1999)

MU Ahmed: "Analysis of human rotavirus G serotype in Bangladesh by enzyme-linked immunosorbent assay and polymerase chain reaction."J.Diarrheal Dis.Res. 17. 22-27 (2000)

MU Ahmed：“通过酶联免疫吸附测定和聚合酶链反应分析孟加拉国人轮状病毒 G 血清型。”J.Diarreal Dis.Res。