TAJIMA Sigeru RIKEN Retrovirus Research Unit,Special post doctoral fellow, 分子ウイルス学研究ユニット, 基礎科学特別研究員 (60311346)
森田 光夫 社団法人家畜改良事業団, 家畜改良技術研究所・遺伝検査部, 課長
森田 裕 チッソ株式会社, 新事業開発室, バイオセンサー部長
|Budget Amount *help
¥14,200,000 (Direct Cost: ¥14,200,000)
Fiscal Year 2002: ¥5,300,000 (Direct Cost: ¥5,300,000)
Fiscal Year 2001: ¥8,900,000 (Direct Cost: ¥8,900,000)
Bovine leukemia virus (BLV) is known as an exogenous retrovirus associated with enzootic bovine leucosis (EBL), which is the most common neoplastic disease of cattle. We investigated the relationship between polymorphism of bovine MHC (BoLA) gene and resistance or susceptibility to BLV-induced lymphoma. The second exon of the BoLA class II DRB3 gene was sequenced for 81 BLV-infected animals with 3 independent stages such as an aleukemic healthy, persistent lymphocytosis and lymphoma. Sequence analysis revealed that alleles encoding Arg or Lys, Glu, Arg and Val at residues 71, 74, 77 and 78 of beta chain of DR might be related with resistance to tumor development.
Next, we developed a more precise method for sequence-based typing (SBT), which allows the identification of specific BoLA-DRB3 alleles in large numbers of animals. Using our SBT methods, we determined the nucleotide sequences of exon 2 of the BoLA-DRB3 alleles of a total of 471 individuals has belonged to four distinct breeds
of catche, namely, Japanese Black, Japanese Shorthorn, Holstein and Jersey. We identified the 34 previously reported alleles and four novel alleles and the frequency of BoLA-DRB3 alleles in each breed.
We have reported previously that the alleles of the ovine leukocyte antigen (OLA)-DRB1 gene that encode the Arg-Lys (RK) motif and the Ser-Arg (SR) motif at positions b^<70/71> of the OLA-DRb1 domain are associated with resistance and susceptibility, respectively, to development of BLV-induced ovine lymphoma. Here, to investigate the different immune response in sheep that carried alleles associated with resistance and susceptible for 30 weeks after infection with BLV, we selected sheep that had the RK/RK or SR/SR genotype among the 52 sheep analyzed by polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing of PCR product for the OLA-DRB1 exon 2 and infected them with BLV. The level of incorporation of [^3H]thymidine by peripheral blood mononuclear cells from the sheep with RK/RK genotype gave a strong response to BLV virion antigen and synthetic antigenic peptides that corresponded to T-helper epitope of the BLV envelope glycoprotein gp51. In contrast, the sheep with SR/SR genotype showed a strong response to BLV virion antigen and synthetic antigenic peptides that corresponded to T-cytotoxic and B-cell epitopes. In such cases, the animals with the RK/RK strongly expressed IFN-g, the animals with SR/SR genotype strongly expressed IL-2. To determine the proliferating cells, we tried a blocking assay with monoclonal antibodies such as anti-CD4, -CD8 and -DR molecule. We found that these proliferating cells were MHC-restricted CD4^+T-cells. Less