ANALYSIS OF INTRACELLULAR ADAPTATION MECHANISMS OF PROTOZOAN PARASITES AND HOST ELIMINATION

本项目旨在阐明寄生虫对宿主细胞环境的适应机制，从而建立细胞内寄生虫感染的控制方法。我们克隆了一个与核苷酸从头合成有关的核糖核苷酸还原酶小亚基（R2）基因。为克隆瑟氏泰勒虫的基因，根据环形泰勒虫的相应基因序列设计了一对寡核苷酸引物进行PCR扩增。由于反转录-PCR产物含有该基因的部分片段，因此进行5 '-3' RACE（cDNA末端的快速扩增）以获得全长mRNA序列。用cDNA探针筛选瑟氏锥虫基因组DNA文库，获得了一个含有R2基因的基因组克隆。基因组DNA克隆的序列分析表明，R2基因含有至少2个内含子。R2 C端氨基酸序列预测结果与宿主R2的C端氨基酸序列差异显著，表明可以设计针对该酶的特异性抑制肽，并对在红细胞期表达的卡巴巴利和马B.用感染的马血清筛选来自B.caballi和B.equi裂殖子的cDNA文库。从马B. equicDNA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，与疟原虫红细胞内期表达的表面分子结构相似。克隆了B.caballi棒状体相关蛋白-1（RAP-1）基因，测定了瑟氏锥虫菌幕蛋白N-末端氨基酸序列，并与已知红细胞蛋白序列进行了比较。由于在数据库搜索中没有发现同源肽，因此推断veil蛋白是寄生虫来源的。

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.