ANALYSIS OF INTRACELLULAR ADAPTATION MECHANISMS OF PROTOZOAN PARASITES AND HOST ELIMINATION

原生动物寄生虫细胞内适应机制分析及宿主消除

• 批准号: 08456143
• 负责人: SUGIMOTO Chihiro
• 金额: $ 4.8万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08456143/
• 关键词: Apicomplexa; Babesia; drug design; erythrocytes; intracellular parasite; parasitism; ribonucleotide reductase; Theileria; 赤血球寄生性原虫; リボヌクレオチド リダクターゼ; 住血原虫; Theileria; メタロプロテアーゼ; リボヌクレオチドリダクターゼ

This project was aiming at clarifying mechanisms of parasite adaptation to host cell environment which will lead us to establish control methods of intracellular parasite infections. We cloned a gene encoding small subunit of ribonucleotide reductase (R2) which is involved denovo synthesis of nucleotides. To clone the gene from Theileria sergenti, oligonucleotide primers for polymerase chain reaction (PCR) were designed based on the corresponding gene sequence of T.annulata. As a reverse transcription-PCR product contained a partial fragment of the gene, 5'-3' RACE (rapid amplification of cDNA ends) were carried out to obtain a full length mRNA sequence. A genomic clone containing R2 gene was also obtained by screening of T.sergenti genomic DNA library with cDNA probe. Sequence analysis of the genomic DNA clone revealed that the R2 gene contains at least 2 introns. Predicted amino acid sequence of R2 C-terminal portion showed significant difference from that of host R2, indicating that inhibitory peptides specific for the parasite enzyme would be designed.Molecules expressed in erythrocytic stage of Babesia caballi and B.equi were also characterized. Libraries of cDNAs from B.caballi and B.equi merozoites were screened with infected horse sera. From B.equi cDNA library, two genes encoding a glutamic acid-rich protein (GARP) and ribosomal protein (L10) were obtained. GARP contained motifs rich in glutamic acid which showed structural similarity to surface molecules expressed in intraerythrocytic stage of malaria parasites. A gene for B.caballi rhoptry-associated protein-l (RAP-1) , one of parasite proteins which plays a central role in parasite invasion into host erythrocytes was also cloned.N-terminal amino acid sequences of veil proteins of T.sergenti were determined and compared to sequences of known erythrocyte proteins. As no homologous poplypeptides were found in data base search, veil proteins were concluded to be parasite-origin.

本项目旨在阐明寄生虫对宿主细胞环境的适应机制，从而建立寄生虫细胞内感染的控制方法。我们克隆了一个编码核糖核苷酸还原酶（R2）小亚基的基因，该基因参与核苷酸的还原合成。为了克隆该基因，根据环孢梭菌相应基因序列设计了聚合酶链反应（PCR）的寡核苷酸引物。由于逆转录- pcr产物含有该基因的部分片段，因此采用5'-3' RACE （cDNA末端快速扩增）获得全长mRNA序列。利用cDNA探针对sergenti t基因组DNA文库进行筛选，获得了含有R2基因的基因组克隆。基因组DNA克隆序列分析显示，R2基因含有至少2个内含子。预测的R2 c端氨基酸序列与宿主R2存在显著差异，表明需要设计出针对该寄生虫酶的抑制肽。对卡巴贝斯虫和马氏巴贝斯虫红细胞期表达的分子也进行了分析。用感染的马血清筛选caballb和马b的分殖子cdna文库。从马羊的cDNA文库中获得了两个编码谷氨酸富蛋白（GARP）和核糖体蛋白（L10）的基因。GARP含有丰富的谷氨酸基序，其结构与疟原虫红细胞内表达的表面分子相似。caballi rhopry -associated protein- 1 （RAP-1）是一种寄生虫蛋白，在寄生虫侵入宿主红细胞的过程中起重要作用。测定了血吸虫面纱蛋白的n端氨基酸序列，并与已知的红细胞蛋白序列进行了比较。由于在数据库检索中未发现同源多肽，因此推断面纱蛋白来源于寄生虫。

项目成果

Nambota A M et al.: "Characterization of some Theileria parva stocks from Zambia using monoclonal antibodies." J.Vet.Med.Sci.59・1. 1-4 (1997)

Nambota A M 等人：“使用单克隆抗体对来自赞比亚的一些泰勒虫种群进行表征。”J.Vet.Med.Sci.59·1 (1997)。

Iwasaki T,Kakuda T,Sako Y,Sugimoto C and Onuma M.: "Differentiation and quantification of Theileria sergenti piroplasm types using type-specific monoclonal antibodies." J.Vet.Med.Sci.(in press).

Iwasaki T、Kakuda T、Sako Y、Sugimoto C 和 Onuma M.：“使用类型特异性单克隆抗体对谢氏泰勒虫梨浆体类型进行区分和定量。”

Kubota S,Kakuda T,Sugimoto C,Waltisbuhl D and Onuma M.: "Allelic analysis of immunodominant major piroplasm surface protein genes of benign Theileria parasites in Australian cattle." J.Protozool.Res.6-1. 13-20 (1996)

Kubota S、Kakuda T、Sugimoto C、Waltisbuhl D 和 Onuma M.：“澳大利亚牛良性泰勒虫寄生虫的免疫显性主要梨质表面蛋白基因的等位基因分析。”

Iwasaki T et al.: "Differentiation and quantification of Theileria piroplasm types using tyoe-specific monoclonal antibodies." J.Vet.Med.Sci.(in press). (1998)

Iwasaki T 等人：“使用 tyoe 特异性单克隆抗体对梨形泰勒虫类型进行区分和定量。”

Kakuda T,Shiki M,Kubota S,Sugimoto C,Brown W C,Chansiri K,Sarataphan N and Onuma M.Molecular: "characterization and phylogeny of benign Theileria species isolated from cattle in Thailand, China and USA based on the major piroplasm surface protein and smal

Kakuda T、Shiki M、Kubota S、Sugimoto C、Brown W C、Chansiri K、Sarataphan N 和 Onuma M.分子：“基于主要梨质表面蛋白从泰国、中国和美国的牛中分离出的良性泰勒虫物种的特征和系统发育