祝！ Kim, J-Y博士誕生

おめでとう、ますますの発展を期待します。

学位論文タイトル
Studies on Comparative Genome Analysis of Theileria Parasites and Vaccine Development against T. orientalis
（Theileria原虫の比較ゲノム解析とT. orientalisに対するワクチン開発に関する研究）


流暢な日本語で横山直明先生の質問に答えるエンさん。


学位論文に関係する主なパブリケーション

(1) Identification of a piroplasm protein of Theileria orientalis that binds to bovine erythrocyte band 3.

Kim, J. Y., Yokoyama, N., Kumar, S., Inoue, N., Inaba, M., Fujisaki, K. and Sugimoto, C.

Theileria orientalis infects cattle and causes various disease symptoms, including anemia and icterus. The erythrocytic stages are responsible for these symptoms but the molecular events involved in these stages have not yet been fully elucidated. In this study, we identified a T. orientalis cDNA that encodes a polypeptide related to identity to the microneme-rhoptry protein of T. parva. Analysis of its recombinant product (ToMRP) by indirect fluorescent-antibody test revealed that it is specifically expressed at the early erythrocytic stage after invasion. This expression disappears during the intermediate stages of intra-erythrocytic development. Its expression then reappears at the late stages after the parasite has divided by binary fission into diad or tetrad forms and before these forms are released from the host erythrocyte. In vitro erythrocyte binding assays showed that ToMRP associates with the Triton-X insoluble fraction of erythrocytes membrane but not with intact erythrocytes. Cosedimentation and western blot analyses revealed that ToMRP binds to band 3, a membrane component of bovine erythrocytes. These observations suggest that ToMRP may be involved in the parasite’s egress from and/or invasion into the host erythrocytes by interacting with a protein in the membrane skeleton of the erythrocyte and thereby modifying the structure and function of the cell.
Mol. Biochem. Parasitol., 2004, 137: 193-200.


(2) Molecular characterization of Theileria orientalis piroplasm protein encoded by an open reading frame (ToORF2) in a genomic fragment.

Kim, J. Y., Yokoyama, N., Kumar, S., Inoue, N., Fujisaki, K. and Sugimoto, C.

In the present study, a novel antigenic protein expressed in the piroplasm stage of Theileria orientalis was characterized. A 4,707 bp genomic fragment amplified by PCR contained two open reading frames (ORFs). The deduced amino acid sequence of the first ORF showed significantly high similarlity to the ubiquitin carboxy terminal hydrolases/proteases while the second ORF (ToORF2) showed homology to several surface antigens of plasmodia. ToORF2 was expressed to determine whether the protein product is expressed by the parasite. In western blot analysis, bovine antiserum from a T. orientalis-infected calf recognized the recombinant protein containing a C-terminal part of the ORF expressed by baculovirus system. Western blot analysis with the anti-ToORF2 mouse serum recognized a 48 kDa protein in T. orientalis piroplasm lysates. Indirect immunofluorescence antibody test by confocal scanning laser microscopic analysis showed that antisera against the recombinant protein recognized T. orientalis piroplasm in the infected erythrocyte. The results from this study indicate that ToORF2 protein is expressed at the piroplasm stage and is immunogenic. This novel antigenic ToORF2 protein could be exploited for vaccine development against bovine piroplasmosis.
J. Vet. Med. Sci., 2004, 66: 957-963.


(3) Molecular Epidemiological Survey of benign Theileria parasites of cattle in Japan: Detection of a new type of major piroplasm surface protein gene.

Kim, J. Y., Yokoyama, N., Kumar, S., Inoue, N., Yamaguchi, T., Sentoku, S., Fujisaki, K. and Sugimoto, C.

Benign Theileria species of cattle are found in most parts of the world.  The major piroplasm surface protein (MPSP), a conserved protein in all Theileria species, has been used as a maker for epidemiological and phylogenetical studies of benign Theileria species.  Parasites with Ikeda- or Chitose-type MPSP genes are dominant in Japan, but we report here mixed infection cases of Theileria parasites with an additional MPSP type parasite infecting cattle in Abashiri District, Hokkaido.  The MPSP gene sequence found in the additional type was closely related to MPSP genes of Theileria parasites found in Southeast Asian countries, including Thailand (Narathiwat) and Indonesia (Java).  Theileria parasites from the blood sample were also distinguishable from the Ikeda or Chitose type parasites by the small subunit (SSU) rRNA gene sequence analysis, and they are grouped into the SSU rRNA types C/D found in Korea, North America, and Spain.  The present finding of mixed infections of cattle with three different types of Theileria makes epidemiological feature of bovine theileriosis in Japan more complex.  We have designed a set of primers specific to this MPSP type in order to conduct further epidemiological study.
J. Vet. Med. Sci., 2004, 66: 251-256.