Structure, function and disease susceptibility of bovine MHC class II molecules

牛白细胞抗原（BoLA） II类基因编码高度多态性的跨膜糖蛋白，该糖蛋白向辅助T细胞提供抗原肽。BoLA-DRB3被认为是功能最重要的，因为它是活跃转录的，超过102个等位基因已被鉴定为该位点。首先，我们开发了基于序列的聚合酶链反应分型技术（PCR-SBT），该技术可以在DNA序列水平上分配DRB3等位基因，并且可以快速筛选大量动物。接下来，我们设计确定BoLA-DRB3等位基因外显子2的核苷酸序列，共419只动物来自4个不同的牛品种，如日本黑牛。采用PCR-SBT方法对日本短刺和泽西短刺进行了分析，并对BoLA-DRB3等位基因的频率、系统发育关系及遗传背景进行了分析。短句来源采用pcr分型方法，对81只白血病病毒（BLV）感染动物的3个独立阶段（亚白血病健康期、持续性淋巴细胞增多症期和淋巴瘤期）的BoLA-DRB3基因2外显子进行了高度多态性测序。共鉴定出23个不同的BoLA-DR83等位基因，其中包括3个新等位基因。BoLA-DRB3^*14011阳性的健康牛种群高于同一等位基因阳性的200个对照病例的比例承载个体。相比之下，BoLA-DRB3^*1601等位基因在PL和淋巴瘤中最常见。序列分析显示，43头感染blv但健康的牛中约56%携带至少一个BoLA-DRB3等位基因，编码Lys^<71>的Arg^<71>, Glu^<74>, Arg^<77>和Val^<78>的DR分子β1结构域，这表明编码KERV和RERV基序的等位基因可能具有抑制肿瘤发生的作用。相比之下，在23头blv感染的淋巴瘤牛中，约70%携带两个等位基因，分别编码Ala^<74>、Thr^<77>和Tyr^<78>，这表明ATY/ATY基因型可能与淋巴瘤易感性有关。因此，这些结果表明等位基因的存在与blv诱导的白血病发生的抗性和易感性相关。接下来，为了证实MHC II类基因多态性与淋巴瘤的关系，我们用BLV感染了28只羊。在16只患淋巴瘤的绵羊中，38%的羊携带了在残基70和71处编码Arg-Lys（RK）的MHC(OLA)-DRB1基因的特异性等位基因，而在12只无症状期的绵羊中，这一比例为83%。此外，我们用BLV包膜糖蛋白gp 51的t辅助抗原肽、t细胞毒性抗原肽和b细胞表位合成抗原肽对携带RK/XX基因型或XX/XX基因型的羊进行免疫，并在最后一次免疫后两周，用BLV感染羊的血液攻击所有羊。携带抗性等位基因的绵羊对混合多肽表现出较高的免疫应答，未发生白血病。少

The bovine leukocyte antigen (BoLA)class II genes code for highly polymorphic transmembrane glycoproteins that present antigenic peptides to helper T cells. The BoLA-DRB3 is thought to be the most functionally significant as it is actively transcribed and in excess of 102 alleles have been characterized for this locus. First, we developed the technique of polymerase chain reaction-sequence based typing(PCR-SBT)which can assign DRB3 alleles in DNA sequence level and would allow fast screening of large numbers of animals. Next, we designed to determine the nucleotide sequences of exon 2 of BoLA-DRB3 alleles in a total of 419 animalks from four distinct cattle breeds such as Japanese Black. Japanese Shorthorn Holstein and Jersey by our PCR-SBT method, and the allele frequencies and the phylogenetic relationship of BoLA-DRB3 alleles and the genetic background of these populations are discussed.The nucleotide sequencing of exon 2 of the BoLA-DRB3 gene, which is highly polymorphic, of 81 bov … More ine leukemia virus(BLV)-infected animals with 3 independent stages such as an aleukemic healthy, persistent lymphocytosis(PL)and lymphoma was determined by PCR-sequenced-based typing. We identified 23 distinct BoLA-DR83 alleles, including 3 new alleles. The population of healthy cattle positive for BoLA-DRB3^*14011 was higher than the proportion-bearing individuals in 200 control cases that were positive for the same allele. By contrast, the BoLA-DRB3^*1601 alleles was found most frequently in cattle with PL and lymphoma. Sequence analysis revealed that, approximately 56 % of 43 BLV-infected but healthy cattle carry at least one BoLA-DRB3 allele encoding Arg^<71> of Lys^<71>, Glu^<74>, Arg^<77> and Val^<78> of β1 domain of DR molecule, which suggested that alleles encoding the KERV and RERV motifs might protect against tumor development. By contrast, approximately 70% of 23 BLV-infected cattle with lymphoma carry two alleles encoding Ala^<74>, Thr^<77> and Tyr^<78>, indicating that the ATY/ATY genotype might be associated with susceptibility to lymphoma. Such, these results suggest that the existence of alleles associated with resistance and susceptibility to BLV-induced leukemogenesis.Next, to confirm the association between polymorphism of MHC class II gene and lymphoma, we infected 28 sheep with BLV.Among the 16 sheep with lymphoma, 38% carried specific alleles of the ovine MHC(OLA)-DRB1 gene encoding Arg-Lys(RK)at residues 70 and 71 as compared to 83% among the 12 sheep at the asymptomatic stage. Furthermore, we immunized sheep carrying either a RK/XX genotype or a XX/XX genotype with a synthetic antigenic peptides of T-helper, T-cytotoxic, and B-cell epitopes of BLV envelope glycoprotein gp 51, and two weeks after the last immunization, all sheep were challenged with blood from a BLV-infected sheep. Sheep carrying resistant allele showed high immunoresponse against the mixed peptides and did not proceeded to leukemia. Less