Suarez C E, Palmer G H, Jasmer D P, Hines S A, Perryman L E, McElwain T F
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman.
Mol Biochem Parasitol. 1991 May;46(1):45-52. doi: 10.1016/0166-6851(91)90197-e.
A clone expressing a surface exposed, conserved epitope of a 60-kDa merozoite polypeptide was identified in a cDNA library constructed from a cloned Mexico strain of Babesia bovis. Sequencing of the 1.9-kb insert (pBv60) revealed an open reading frame encoding a 65-kDa polypeptide with a signal peptide and a tandemly repeated region. Monoclonal antibody 23/56.156, which binds a surface exposed epitope on the native polypeptide, specifically immunoprecipitated [35S]methionine-labeled polypeptides ranging from 60-30 kDa from pBv60 directed transcription and translation. Antibodies raised in rabbits against recombinant polypeptide reacted with the live merozoite surface in a polar immunofluorescence pattern, immunoprecipitated the native 60-kDa polypeptide, and were used to deplete the polypeptide by adsorption from a preparation of native [35S]methionine-labeled merozoite antigen. Restriction enzyme analysis indicated a single gene copy and the absence of introns. Hybridization demonstrated the presence of the gene in Mexico, Australia 'L', and Texas strains of B. bovis, but not in Babesia bigemina. A slightly different hybridization pattern was present in uncloned Australia 'L' B. bovis, indicating sequence diversity in the Bv60 gene among isolates. Cloning and structural analysis of pBv60 provides a source of defined antigen for determining the role of conserved merozoite surface epitopes in protective immunity against babesiosis.
在从牛巴贝斯虫墨西哥克隆株构建的cDNA文库中，鉴定出一个表达60 kDa裂殖子多肽表面暴露保守表位的克隆。对1.9 kb插入片段（pBv60）进行测序，发现一个开放阅读框，编码一个带有信号肽和串联重复区域的65 kDa多肽。单克隆抗体23/56.156可结合天然多肽上的表面暴露表位，它特异性免疫沉淀了来自pBv60定向转录和翻译的、大小从60 kDa到30 kDa的[35S]甲硫氨酸标记多肽。用重组多肽免疫兔产生的抗体，以极性免疫荧光模式与活裂殖子表面发生反应，免疫沉淀天然的60 kDa多肽，并用于从天然[35S]甲硫氨酸标记裂殖子抗原制剂中通过吸附去除该多肽。限制性内切酶分析表明存在单基因拷贝且无内含子。杂交证明该基因存在于牛巴贝斯虫的墨西哥株、澳大利亚“L”株和得克萨斯株中，但不存在于双芽巴贝斯虫中。未克隆的澳大利亚“L”株牛巴贝斯虫呈现出略有不同的杂交模式，表明不同分离株中Bv60基因存在序列多样性。pBv60的克隆和结构分析为确定保守裂殖子表面表位在抗巴贝斯虫病保护性免疫中的作用提供了一个明确的抗原来源。
