Research on the expression of dengue virus gene.

登革热病毒基因表达研究.

• 批准号: 63570213
• 负责人: MAKINO Yoshihiro
• 金额: $ 1.28万
• 依托单位: University of the Ryukyus
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1989
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63570213/
• 关键词: Dengue 4 virus; Gene expression; Baculovirus; dengue core gene; dengue envelope gene; デングウイルス; コア遺伝子; 核内抗原

1. The dengue 4 virus (DEN-4) core gene was inserted into the baculovirus polyhedrin gene region and obtained a recombinant baculovirus (373-core), which directed the synthesis of a DEN-4 core fusion protein. The Sf-9 cells infected with the recombinant virus and incubated for 24-48hr were fixed and stained by peroxidase anti-peroxidase(PAP)staining method using DEN patient sera. The results showed that the patient sera did not react with the recombinant DEN-4 core protein, It can be explained as follows: (1) Only a portion of the epitopes on the authentic DEN-4 core protein may be present in a recombinant core protein. Thus only a part of the core antibodies in the patient sera reacted with the recombinant core protein. (2) The DEN core protein does not migrate to the surface of the infected cells. Therefore, the core protein is not recognized by immune mechanism, thus little DEN core antibody are produced.2. The recombinant baculovirus which contained DEN-4 envelope(E) gene was constructed. This recombinant baculovirus (401E) directed the synthesis of DEN-4 envelope fusion protein with a molecular weight of about 43K dalton. The recombinant virus infected cells reacted with DEN patient sera as well as DEN specific polyclonal and monoclonal antibody as examined by PAP staining method. The staining titers of the patient sera were well correlated with ELISA titers. Thus the usefulness of the recombinant E protein as a diagnostic antigen has been suggested.

1.将登革4型病毒（DEN-4）的核心基因插入杆状病毒多角体蛋白基因区，获得重组杆状病毒（373-core），指导DEN-4核心融合蛋白的合成。用重组病毒感染并孵育24- 48小时的Sf-9细胞固定，并使用DEN患者血清通过过氧化物酶抗过氧化物酶（PAP）染色法染色。结果表明，患者血清与重组DEN-4核心蛋白不发生反应，这可能是由于：（1）重组核心蛋白中可能只含有DEN-4核心蛋白上的部分抗原决定簇。因此，患者血清中只有一部分核心抗体与重组核心蛋白反应。(2)DEN核心蛋白不会迁移到受感染细胞的表面。因此，核心蛋白不被免疫机制所识别，因此DEN核心抗体的产生很少.构建了含DEN-4 E基因的重组杆状病毒。该重组杆状病毒（401 E）定向合成了分子量约为43 K道尔顿的DEN-4包膜融合蛋白。PAP染色法检测重组病毒感染细胞与DEN患者血清及DEN特异性多克隆抗体和单克隆抗体反应。患者血清的染色滴度与ELISA滴度具有良好的相关性。因此，重组E蛋白作为诊断抗原的有用性已被提出。

项目成果

Masayuki Tadano: "Detection of Dengue 4 Virus Core Protein in the Nucleus.I.A Monoclonal Antibody to Dengue 4 Virus Reacts with the Antigen in the Nucleus and Cytoplasm" J.gen.Virol.70. 1409-1415 (1989)

Masayuki Tadano：“检测细胞核中的登革热 4 病毒核心蛋白。I.A 单克隆抗体针对登革热 4 病毒与细胞核和细胞质中的抗原发生反应”J.gen.Virol.70。

Yoshihiro Makino: "Detection of Dengue 4 Virus Core Protein in the Nucleus.II.Antibody against Dengue 4 Core Protein Produced by a Recombinant Baculovirus Reacts with the Antigen in the Nucleus" J.gen.Virol.70. 1417-1425 (1989)

Yoshihiro Makino：“细胞核中登革热 4 病毒核心蛋白的检测。II.重组杆状病毒产生的针对登革热 4 核心蛋白的抗体与细胞核中的抗原反应”J.gen.Virol.70。

Yoshihiro Makino.: Journal of general Virology.

Yoshihiro Makino.：普通病毒学杂志。

Masayuki Tadano: "Detection of Dengue 4 Virus Core Protein in the Nucleus. I. A Monoclonal antibody to Dengue 4 virus Reacts with the Antigen in the Nucleus and Cytoplasm" J. gen. Virol.70. 1409-1415 (1989)

Masayuki Tadano：“细胞核中登革热 4 病毒核心蛋白的检测。I. 登革热 4 病毒单克隆抗体与细胞核和细胞质中的抗原发生反应”J. gen。

Yoshihiro Makino: "Detection of Dengue 4 Virus Core Protein in the Nucleus. II. Antibody against Dengue 4 Core Protein Produced by a Recombinant Baculovirus Reacts with the Antigen in the Nucleus" J. gen. Virol.70. 1417-1425 (1989)

Yoshihiro Makino：“细胞核中登革热 4 病毒核心蛋白的检测。II.重组杆状病毒产生的抗登革热 4 核心蛋白的抗体与细胞核中的抗原反应”J. gen。