Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA, USA 99164-7040.
Animal Disease Research Unit, Agricultural Research Service, United States Department of Agriculture, Pullman, WA, USA 99164-6630.
Parasitology. 2011 Jun;138(7):809-18. doi: 10.1017/S0031182011000321. Epub 2011 Apr 18.
The Babesia bovis genome encodes a rap-1 related gene denominated RAP-1 related antigen (RRA). In this study, we analysed the pattern of expression, immunogenicity and functional relevance of RRA.
Phylogenetic analysis was performed using the program Phylip. Expression of rra was analysed by Northern blots, RT-PCR, immunoprecipitation, Western blots and immunofluorescence. RRA antigenicity was tested by T-cell proliferation and Western blot analysis, and functional relevance was determined in an in vitro neutralization assay.
RRA is more closely related to RAP-1b of Babesia bigemina than to B. bovis RAP-1, and it is highly conserved among distinct strains. Transcriptional analysis suggests lower numbers of rra transcripts compared to rap-1. Immunoprecipitation of metabolically labelled B. bovis proteins with antibodies against synthetic peptides representing predicted antigenic regions of RRA confirmed the expression of a ∼43 kDa RRA in cultured merozoites. Antibodies present in B. bovis hyperimmune sera, but not in field-infected cattle sera, reacted weakly with recombinant RRA, and no significant stimulation was obtained using recombinant RRA as antigen in T-cell proliferation assays, indicating that RRA is a subdominant antigen. Antibodies against RRA synthetic peptides reacted with merozoites using immunofluorescence, and were able to significantly inhibit erythrocyte invasion in in vitro neutralization tests, suggesting functional relevance for parasite survival.
B. bovis express a novel subdominant RAP-1-like molecule that may contribute to erythrocyte invasion and/or egression by the parasite.
牛巴贝斯虫基因组编码一种 rap-1 相关基因，命名为 rap-1 相关抗原（RRA）。本研究分析了 RRA 的表达模式、免疫原性和功能相关性。
使用 Phylip 程序进行系统发育分析。通过 Northern blot、RT-PCR、免疫沉淀、Western blot 和免疫荧光分析 rra 的表达。通过 T 细胞增殖和 Western blot 分析检测 RRA 抗原性，并在体外中和试验中确定其功能相关性。
RRA 与双芽巴贝斯虫的 RAP-1b 比与牛巴贝斯虫的 RAP-1 更为密切相关，并且在不同株之间高度保守。转录分析表明 rra 的转录本数量比 rap-1 少。用针对 RRA 预测抗原区域的合成肽的抗体免疫沉淀代谢标记的牛巴贝斯虫蛋白，证实了在培养的裂殖子中表达了一个约 43 kDa 的 RRA。牛巴贝斯虫高免血清中存在的抗体，但在田间感染牛血清中不存在的抗体，与重组 RRA 反应较弱，并且在 T 细胞增殖试验中使用重组 RRA 作为抗原未获得显著刺激，表明 RRA 是一个亚显性抗原。针对 RRA 合成肽的抗体通过免疫荧光与裂殖子反应，并能够在体外中和试验中显著抑制红细胞入侵，表明其对寄生虫生存具有功能相关性。
牛巴贝斯虫表达一种新的亚显性 rap-1 样分子，可能有助于寄生虫对红细胞的入侵和/或逸出。
