Takeshima S, Nakai Y, Ohta M, Aida Y
Retrovirus Research Unit, RIKEN, Wako, Saitama, Japan.
J Dairy Sci. 2002 Jun;85(6):1630-2. doi: 10.3168/jds.S0022-0302(02)74234-3.
A study was made of exon 2 of the bovine leukocyte antigen BoLA-DRB3 gene of 176 Japanese Shorthorn cattle at six farms in Japan using polymerase chain reaction-sequence-based typing (PCR-SBT). An initial round of PCR using conserved locus-specific primers, a second round of PCR using a locus-specific primer, and at least one sequence-specific primer (SSP), followed by direct sequencing of products of PCR with SSP were conducted. Twenty-one BoLA-DRB3 alleles were identified with frequencies ranging from 0.3 to 19.6% in 176 individuals, and two of these alleles were new alleles that have not been reported previously. The three most frequently observed alleles (DRB3*1201, *0301, and *0801) accounted for 43.8% of the alleles in the population of these herds. Next, we tested the products of amplification by PCR of BoLA-DRB3 exon 2 with RsaI, BstYI, and HaeIII, and identified 18 previously described PCR-restriction fragment length polymorphism (RFLP) types. The PCR-RFLP types reflected the results of PCR-SBT exactly. Our results indicate that exon 2 of the BoLA-DRB3 gene is highly polymorphic in Japanese Shorthorn cattle.
利用基于聚合酶链反应-序列分型(PCR-SBT)技术,对日本六个农场的176头日本短角牛的牛白细胞抗原BoLA-DRB3基因的外显子2进行了研究。首先使用保守的位点特异性引物进行第一轮PCR,然后使用位点特异性引物和至少一种序列特异性引物(SSP)进行第二轮PCR,接着对SSP-PCR产物进行直接测序。在176个个体中鉴定出21个BoLA-DRB3等位基因,其频率范围为0.3%至19.6%,其中两个等位基因为先前未报道的新等位基因。三个最常见的等位基因(DRB3*1201、0301和0801)占这些牛群群体中等位基因的43.8%。接下来,我们用RsaI、BstYI和HaeIII对BoLA-DRB3外显子2的PCR扩增产物进行检测,鉴定出18种先前描述的PCR-限制性片段长度多态性(RFLP)类型。PCR-RFLP类型与PCR-SBT结果完全相符。我们的结果表明,BoLA-DRB3基因的外显子2在日本短角牛中具有高度多态性。