State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, Hubei, China.
Key Laboratory of Preventive Veterinary Medicine in Hubei Province, Wuhan, 430070, Hubei, China.
Parasit Vectors. 2018 Mar 27;11(1):205. doi: 10.1186/s13071-018-2795-7.
The spherical body is a distinct organelle only existing in Babesia and Theileria. Spherical body proteins (SBPs) are secreted from spherical bodies and incorporated into the cytoplasm of infected erythrocytes during invasion and post-invasion stages. Four different SBP homologues (SBP1, SBP2, SBP3 and SBP4) have been identified in Babesia bovis and Babesia bigemina. So far, there has been no report available about the identification of SBPs in Babesia orientalis.
The SBP3-like in B. orientalis (BoSBP3-like) was cloned, sequenced, characterized and compared to the SBP3 sequences of B. bovis and B. bigemina by bioinformatics analyses. The BoSBP3-like gene was truncated into three fragments: BoSBP3-like-1 (915 bp), BoSBP3-like-2 (1311 bp) and BoSBP3-like-3 (1011 bp), which were amplified and cloned into the expression vector pET-28a and expressed as three truncated recombinant (His-fusion) proteins. The immunogenicity, native forms and localization of BoSBP3-like were identified by western blot and indirect immunofluorescence assay (IFA).
The BoSBP3-like gene was intronless with an open reading frame (ORF) of 3237 bp, encoded a 1079 amino acid polypeptide with a predicted size of 135 kDa, and contained a cysteine-rich region, three dispersing FAINT domains and a signal peptide (1-16 aa) at the N-terminus. The amino acid sequence of BoSBP3-like was 61.6 and 35.0% identical to that of B. bovis and B. bigemina, respectively. BoSBP3-like was identified as 135 kDa in the parasite lysate by rabbit antiserum against the truncated recombinant BoSBP3-like-1 (rBoSBP3-like-1). Three specific bands corresponding to rBoSBP3-like-1 (1-305 aa, 43 kDa), rBoSBP3-like-2 (306-742 aa, 58 kDa) and rBoSBP3-like-3 (743-1079 aa, 52 kDa) were detected by reaction with serum from B. orientalis-infected buffalo. The BoSBP3-like was not only localized in the spherical body of B. orientalis but also in the cytoplasm of infected erythrocytes of buffalo as puncta-like protein specks at both single and paired parasite development stages.
Through secretion into the cytoplasm of infected erythrocytes, BoSBP3-like may play a significant role in adaptation, interaction, and modification related to the host environment to benefit the growth and survival of Babesia. BoSBP3-like could react with the serum from B. orientalis-infected buffalo, but not healthy buffalo, implicating that BoSBP3-like is highly antigenic and may serve as a candidate diagnostic antigen for the detection of B. orientalis.
球体是仅存在于巴贝西虫属和泰勒虫属中的独特细胞器。球体蛋白（SBPs）从球体中分泌出来，并在入侵和入侵后阶段整合到感染的红细胞细胞质中。已经在牛巴贝斯虫和双芽巴贝斯虫中鉴定出四种不同的 SBP 同源物（SBP1、SBP2、SBP3 和 SBP4）。到目前为止，还没有关于东方巴贝斯虫中 SBPs 鉴定的报道。
克隆、测序、表征东方巴贝斯虫中的 SBP3 样蛋白（BoSBP3-like），并通过生物信息学分析与牛巴贝斯虫和双芽巴贝斯虫的 SBP3 序列进行比较。BoSBP3-like 基因被截断成三个片段：BoSBP3-like-1（915 bp）、BoSBP3-like-2（1311 bp）和 BoSBP3-like-3（1011 bp），并扩增和克隆到表达载体 pET-28a 中，并表达为三个截断的重组（His 融合）蛋白。通过 Western blot 和间接免疫荧光分析（IFA）鉴定 BoSBP3-like 的免疫原性、天然形式和定位。
BoSBP3-like 基因无内含子，开放阅读框（ORF）为 3237 bp，编码 1079 个氨基酸的多肽，预测大小为 135 kDa，含有富含半胱氨酸的区域、三个分散的 FAINT 结构域和 N 端的信号肽（1-16 aa）。BoSBP3-like 的氨基酸序列与牛巴贝斯虫和双芽巴贝斯虫分别有 61.6%和 35.0%的同一性。兔抗截短重组 BoSBP3-like-1（rBoSBP3-like-1）的抗血清在寄生虫裂解物中鉴定出 135 kDa 的 BoSBP3-like。与东方巴贝斯虫感染水牛的血清反应检测到三个与 rBoSBP3-like-1（1-305 aa，43 kDa）、rBoSBP3-like-2（306-742 aa，58 kDa）和 rBoSBP3-like-3（743-1079 aa，52 kDa）相对应的特异性条带。BoSBP3-like 不仅定位于东方巴贝斯虫的球体中，而且还定位于感染水牛红细胞的细胞质中，在单个和成对寄生虫发育阶段呈点状蛋白斑点。
通过分泌到感染的红细胞细胞质中，BoSBP3-like 可能在与宿主环境相关的适应、相互作用和修饰中发挥重要作用，有利于巴贝斯虫的生长和存活。BoSBP3-like 可与东方巴贝斯虫感染水牛的血清反应，但与健康水牛的血清不反应，表明 BoSBP3-like 具有高度抗原性，可能是检测东方巴贝斯虫的候选诊断抗原。
