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.

该项目旨在阐明寄生虫适应宿主细胞环境的机制，这将使我们建立细胞内寄生虫感染的控制方法。我们克隆了一个编码核糖核苷酸还原酶（R2）的小亚基的基因，该基因涉及核苷酸的Denovo合成。为了克隆来自Theileria sergenti的基因，基于T.annulata的相应基因序列设计了用于聚合酶链反应（PCR）的寡核苷酸引物。由于逆转录PCR产物包含基因的部分片段，因此进行了5'-3'种族（cDNA末端的快速扩增）以获得全长mRNA序列。还通过用cDNA探针筛选T.Sergenti基因组DNA文库获得了含有R2基因的基因组克隆。基因组DNA克隆的序列分析表明，R2基因至少包含2个内含子。预测的R2 C末端部分的氨基酸序列与宿主R2的氨基酸序列显示出显着差异，表明将设计针对寄生虫酶的抑制性肽。用受感染的马血清筛选了B. caballi和B.Equi Merozoites的CDNA库。从B.Equi cDNA文库中，获得了两个编码富含谷氨酸蛋白（GARP）和核糖体蛋白（L10）的基因。 GARP含有富含谷氨酸的基序，这些基序与疟原虫内肉体内表达的表面分子相似。克隆也克隆了b.caballi Rhoptry相关蛋白L（RAP-1）的基因，是寄生虫蛋白之一，它在寄生虫入侵寄生于宿主红细胞中起着核心作用。N-末端氨基酸t.ssergenti的叶子氨基酸序列被确定，并与已知的伊氏蛋白质蛋白的序列进行比较。由于在数据库搜索中没有发现同源的杂种肽，因此透露面纱蛋白是寄生虫 - 蛋白质。

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