The Model Study of Vaccine Development against Cysticercosis by Expression-Library Immunization using Taenia taeniaeformis-rat system

利用带绦虫-大鼠系统表达文库免疫开发囊尾蚴病疫苗的模型研究

• 批准号: 12680811
• 负责人: OKAMOTO Munehiro
• 金额: $ 2.3万
• 依托单位: Tottori University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680811/
• 关键词: Taenia taeniaeformis; Library Vaccine; rat; Protective Antigen; immunity; Taeniid Cestode; テニア科条虫; ネコ系虫; 防御、抗原遺伝子

Cysticercosis, one of the most serious larval cestodiasis of medical importance in humans and of economic importance in pigs, is spreading worldwide, especially in Asia, Africa and Latin America. The trial of the vaccine development against Taenia solium cysticercosis has been made in many countries, and actually some candidate molecules have been also reported. However, the effects of vaccine used those molecules have been not always perfect. Expression-library immunization has been, proposed as an effective means to screen a large number of genes of the pathogen as candidate protective molecules. In this study, we examined the efficacy of expression-library immunization for vaccine development against T.solium cysticercosis using T.taeniaeformis-rat model systemTotal RNAs were isolated from last 10 segments of adult T.taeniaeformis by Isogen LS, and poly A RNA was purified using oligotex-dT30 (Takara). cDNA library was produced using SuperScript Plasmid System (Inbitorogen), which contain a mammalian expression vector, pCMV・SPORT6. Transformed E.coli were plated onto LB-plate containing ampicillin, and the cells that grew on the plate were resuspended in TYGPN nedium. Each clone was sequenced and classified with the property of the insert. Twenty to thirty independent clones were simultaneously propagated in LB medium with ampicillin, and plasmid DNAs were isolated and purified using End Free Plasmid Kit. (QIAGEN). For the immunization, purified plasmid DNAs were injected directly into the spleen and muscle of miceIt was reported that the number of the mitochondrion in adult taeniid cestode was not so many. However, more than 30% of the sequenced clones had mitochondrial genes. Specific antibody to Taenia taeniaeformis was detected by western-blot in the mouse immunized with Library-Vaccine

囊尾蚴病是最严重的幼虫绦虫病之一，对人类具有医学重要性，对猪具有经济重要性，正在全世界蔓延，特别是在亚洲、非洲和拉丁美洲。许多国家都进行了猪带绦虫囊尾蚴病疫苗的研制试验，实际上也报道了一些候选分子。然而，使用这些分子的疫苗的效果并不总是完美的。表达文库免疫已被认为是筛选大量病原体基因作为候选保护分子的有效手段。在本研究中，我们使用 T.taeniaeformis 大鼠模型系统检查了表达文库免疫用于开发针对 T.solium 囊尾蚴病疫苗的功效。通过 Isogen LS 从成年 T.taeniaeformis 的最后 10 个片段中分离出总 RNA，并使用oligotex-dT30 (Takara) 纯化聚 A RNA。 cDNA文库是使用SuperScript Plasmid System (Inbitorogen)制作的，其中含有哺乳动物表达载体pCMV·SPORT6。将转化的大肠杆菌接种到含有氨苄青霉素的LB平板上，并将在平板上生长的细胞重悬于TYGPN培养基中。对每个克隆进行测序并根据插入片段的特性进行分类。 20至30个独立克隆同时在含有氨苄青霉素的LB培养基中增殖，并使用End Free Plasmid Kit分离和纯化质粒DNA。 （凯杰）。为了进行免疫，将纯化的质粒DNA直接注射到小鼠的脾脏和肌肉中。据报道，成年带绦虫的线粒体数量并不多。然而，超过 30% 的测序克隆具有线粒体基因。在用文库疫苗免疫的小鼠中通过蛋白质印迹检测到带绦虫特异性抗体

项目成果

Yamasaki H., Nakano M., et al.: "DNA differential diagnosis of human taeniid cestodes by base excision sequence scanning thymine-base reader analysis with mitochondrial genes"Journal of Clinical Microbiology. 40. 3818-3821 (2002)

Yamasaki H.、Nakano M.等人：“通过碱基切除序列扫描胸腺嘧啶碱基阅读器分析与线粒体基因对人绦虫绦虫进行DNA鉴别诊断”临床微生物学杂志。

Ito A.et al.: "Multiple genotypes of Taenia solium ? ramifications for control"Acta Tropica. (in press). (2003)

Ito A.等人：“猪带绦虫的多种基因型？控制的后果”Acta Tropica。

Nakao M., Okamoto M., et al.: "A phylogenetic hypothesis for the distribution of two genotypes of the pig tapeworm Taenia solium worldwide"Parasitology. 124. 657-662 (2002)

Nakao M.、Okamoto M. 等人：“全球猪绦虫带绦虫两种基因型分布的系统发育假说”寄生虫学。

Ito A., Yamasaki H., Nakao M., Sako Y., Okamoto M.et al.: "Multiple genotypes of Taenia solium-ramifications for diagnosis, treatment and control"Acta Tropica. 87. 95-101 (2003)

Ito A.、Yamasaki H.、Nakao M.、Sako Y.、Okamoto M.等人：“猪带绦虫的多种基因型对诊断、治疗和控制的影响”《热带学报》。

Nakao M. et al.: "A phylogenetic hypothesis for the distribution of two genotypes of the pig tapeworm Taenia solium worldwide"Parasitology. 124・6. 657-662 (2002)

Nakao M. 等：“猪带绦虫世界范围内两种基因型分布的系统发育假说”，《寄生虫学》124・6（2002 年）。