Detection of bovine MHC genes associated with resistance or susceptibility to bovine leukemia virus-induced lymphoma

牛MHC基因的检测与牛白血病病毒诱导的淋巴瘤的抗性或易感性相关

• 批准号: 13556055
• 负责人: AIDA Yoko
• 金额: $ 9.09万
• 依托单位: RIKEN
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13556055/
• 关键词: Bovine MHC (BoLA)-DRB3 exon 2; Bovine leukemia virus (BLV); Polymorphism; Ovine MHC (OLA)-DRB1; PCR-Sequence Based Typing; PCR-RFLP method; Disease susceptibility; BLV-induced lymphoma; BLV Env gp51抗原; モノクローナル抗体; PCR-Sequence Based Typing法; ウシ主

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 … More 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

牛白血病病毒(BLV)是一种与流行性牛白血病(EBL)相关的外源性逆转录病毒，是牛最常见的肿瘤性疾病。我们研究了牛MHC(BOLA)基因多态性与BLV诱导的淋巴瘤抗性或易感性的关系。对81只BLV感染动物的BolaII类DRB3基因外显子第二外显子进行了测序，包括白血病、持续性淋巴细胞增多症和淋巴瘤3个独立阶段。序列分析表明，编码DRβ链第71、74、77和78位残基的Arg或Lys、Glu、Arg和Val等位基因可能与肿瘤的抗性有关。接下来，我们发展了一种更精确的基于序列的分型方法(SBT)，可以在大量动物中识别特定的BOLA-DRB3等位基因。用我们的sbt方法测定了4个不同品种…的boA-dr3等位基因外显子2的核苷酸序列。更多的是甜点，即日本黑、日本短刺、荷斯坦和泽西。我们鉴定了已报道的34个等位基因和4个新的等位基因以及BoLA-DRB3等位基因在每个品种中的频率。我们先前已经报道，编码Arg-Lys(RK)基序的绵羊白细胞抗原(Ola)-DRB1基因的等位基因和Ola-DRB1结构域b^&lt；70/71&gt；的Ser-Arg(SR)基序的等位基因分别与BLV诱导的绵羊淋巴瘤的抗性和易感性有关。为了研究携带BLV抗性相关等位基因和易感等位基因的绵羊感染BLV后30周的不同免疫反应，我们从52只绵羊中筛选出RK/RK或SR/SR两种基因型的绵羊，通过聚合酶链式反应-限制性片段长度多态性和DNA测序分析Ola-DRB1外显子2的DNA序列，并将其感染BLV。RK/RK基因型绵羊外周血单个核细胞对BLV病毒粒子抗原和与BLV囊膜糖蛋白gp51的T辅助表位相对应的合成抗原肽有较强的[~(3 H)]胸腺嘧啶核苷掺入水平。相反，SR/SR基因的绵羊对BLV病毒粒子抗原和合成的T细胞毒性和B细胞表位的抗原肽表现出强烈的反应。在这种情况下，RK/RK的动物强烈表达干扰素-g，SR/SR基因的动物强烈表达IL-2。为了确定增殖细胞，我们尝试了单抗阻断试验，如抗CD4、-CD8和-DR分子。我们发现这些增殖细胞是MHC限制性的CD4^+T细胞。较少

项目成果

Tajima S., et al.: "Mutant Tax protein from bovine leukemia virus with enhanced ability to activate the expression of c-fos"J.Virol.. 76. 2557-2562 (2002)

Tajima S.等人：“来自牛白血病病毒的突变Tax蛋白具有增强的激活c-fos表达的能力”J.Virol.. 76. 2557-2562 (2002)

Takeshima S.m et al.: "Characterization of DRB3 alleles in the MHC of Japanese Shorthorn cattle by polymerase chain reaction-sequence-based typing"J.Dairy Sci.. 85. 1630-1632 (2002)

Takeshima S.m 等：“通过聚合酶链反应-基于序列的分型来表征日本短角牛 MHC 中的 DRB3 等位基因”J.Dairy Sci.. 85. 1630-1632 (2002)

Tajima S., Tsukamoto M. and Aida Y.: "Latency of viral expression in vivo is not related to CpG methylation in the U3 and part of the R region of the long teminal repeart of bovine leukemia virus"J.Virol. in press.

Tajima S.、Tsukamoto M. 和 Aida Y.：“体内病毒表达潜伏期与牛白血病病毒长末端重复区 U3 和部分 R 区的 CpG 甲基化无关”J.Virol。

Takeshima S., Nakai Y., Ohta M. and Aida Y.: "Tiping method of bovine major histocompatibility complex"Bulletin of the experimental Farm, Tohoku University. 17:. 19-28 (2001)

Takeshima S.，Nakai Y.，Ohta M.和Aida Y.：“牛主要组织相容性复合体的Tiping方法”东北大学实验农场公告。

Takeshima, S., et al.: "Characterization of DRB3 alleles in the MHC of Japanese Shorthorn cattle by polymerase chain reaction-sequence-based typing"J.Dairy Science. 85. 1630-1632 (2002)

Takeshima, S., et al.：“通过聚合酶链式反应序列分型来表征日本短角牛 MHC 中的 DRB3 等位基因”J.Dairy Science。