Joosten I, Teale A J, van der Poel A, Hensen E J
Department of Immunology, Faculty of Veterinary Medicine, University of Utrecht, The Netherlands.
Anim Genet. 1992;23(2):113-23. doi: 10.1111/j.1365-2052.1992.tb00030.x.
For a long time, the bovine major histocompatibility complex (MHC) (BoLA) class I region was characterized, rather uniquely among mammalian species, as having one expressed locus. Recent reports have suggested otherwise. Selective immunoprecipitation and molecular characterization of products enable a decisive answer to the question of whether there is indeed more than one locus expressed. Therefore, we characterized serologically defined w10 encoding haplotypes in European and African cattle by immunoprecipitation of [35S]-methionine-labelled peripheral blood mononuclear cells (PBMC), followed by one- and two-dimensional isoelectric focusing (1D/2D-IEF) of cell lysates. Monoclonal antibodies (mAb) used were directed against either human class I monomorphic determinants (W6/32 and B1.1G6) or bovine polymorphic determinants expressed on products encoded by serologically defined w10 encoding haplotypes of Boran and Friesian cattle. Sequential immunoprecipitations with W6/32 and B1.1G6 using lysates of PBMC of British Friesian cattle, revealed that from this haplotype W6/32 precipitated one product, whereas B1.1G6 precipitated two products. The product precipitated in addition appeared to be the one that was selectively precipitated by the mAb directed against polymorphic determinants on a product of w10 encoding haplotypes. Additionally, peptide maps of protease V8-digested precipitates showed that this particular 'w10' associated product was distinctly different from the product recognized by W6/32. Thus, we suggest that the two products are distinct gene products and that the product with higher pI is associated with the serologically defined A-locus product, whereas the product with lower pI is the putative second locus product. In the African Boran breed, variants of the serologically defined w10 specificity were found on the basis of IEF typing. These variants appeared to be associated with different second locus products. Therefore, we conclude that serologically defined w10 encoding haplotypes encode at least two independent class I locus products, expressed on normal bovine PBMC. In IEF analysis the additional use of mAb recognizing polymorphic determinants on serologically defined A-locus products highly facilitated the detection and typing of second locus products.
长期以来，牛主要组织相容性复合体（MHC）（BoLA）I类区域的特征是，在哺乳动物物种中相当独特地只有一个表达位点。然而，最近的报告却提出了不同的观点。对产物进行选择性免疫沉淀和分子表征，能够对是否确实存在多个表达位点的问题给出决定性的答案。因此，我们通过对[35S] - 甲硫氨酸标记的外周血单个核细胞（PBMC）进行免疫沉淀，随后对细胞裂解物进行一维及二维等电聚焦（1D/2D - IEF），对欧洲和非洲牛中血清学定义的w10编码单倍型进行了表征。所使用的单克隆抗体（mAb）针对的是人I类单态决定簇（W6/32和B1.1G6）或在血清学定义的博拉牛和弗里斯兰牛w10编码单倍型所编码产物上表达的牛多态决定簇。使用英国弗里斯兰牛PBMC裂解物，先后用W6/32和B1.1G6进行免疫沉淀，结果显示，从该单倍型中，W6/32沉淀出一种产物，而B1.1G6沉淀出两种产物。另外沉淀出的产物似乎就是由针对w10编码单倍型产物上多态决定簇的mAb选择性沉淀的那种产物。此外，蛋白酶V8消化沉淀产物的肽图谱显示，这种特定的“w10”相关产物与W6/32识别的产物明显不同。因此，我们认为这两种产物是不同的基因产物，较高pI的产物与血清学定义的A位点产物相关，而较低pI的产物是假定的第二个位点产物。在非洲博拉品种中，基于IEF分型发现了血清学定义的w10特异性变体。这些变体似乎与不同的第二个位点产物相关。因此，我们得出结论，血清学定义的w10编码单倍型编码至少两种独立的I类位点产物，在正常牛PBMC上表达。在IEF分析中，额外使用识别血清学定义的A位点产物上多态决定簇的mAb极大地促进了第二个位点产物的检测和分型。
