从东方巴贝斯虫中鉴定出一种新型的血小板反应蛋白相关的未知蛋白（BoTRAP2）。
Identification of a novel thrombospondin-related anonymous protein (BoTRAP2) from Babesia orientalis.
机构信息
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.
出版信息
Parasit Vectors. 2019 May 3;12(1):200. doi: 10.1186/s13071-019-3457-0.
BACKGROUND
The thrombospondin-related anonymous protein (TRAP) was first discovered in the sporozoite of Plasmodium falciparum and TRAP family proteins are secreted by micronemes and transported to the parasite surface to participate in the invasion process. Various TRAP proteins have been identified in apicomplexan protozoans, but there have been few reports about TRAP proteins in Babesia orientalis.
METHODS
The functional domain of TRAP2 in B. orientalis was cloned, sequenced, characterized and compared to the TRAP sequences of related apicomplexan parasites. The functional domain of BoTRAP2 was truncated, named BoTRAP2-1, and then cloned into the pET-28a expression vector. Rabbit anti-rBoTRAP2-1 polyclonal antibody was produced by immunizing three rabbits. Western blot analysis was used to identify the native form and immunogenicity of BoTRAP2. The localization of BoTRAP2 was identified by indirect fluorescence assay (IFA).
RESULTS
The amplified genes of BoTRAP2 are 2817 bp in length, encoding a functional domain of about 938 aa with two vWFA domains, one TSP domain and one transmembrane domain. The amino acid sequence of BoTRAP2 has a high similarity with that of B. bovis and B. gibsoni. The predicted tertiary structure of truncated BoTRAP2-1 confirmed that BoTRAP2 contains two vWFA domains and a TSP domain, the main functional areas of the protein. The native BoTRAP2 was identified from B. orientalis lysate by using rabbit polyclonal anti-rBoTRAP2-1. A band corresponding to rBoTRAP2-1 was detected by reaction with serum from a B. orientalis-infected water buffalo, indicating that the protein has a high immunogenicity. IFA showed that BoTRAP2 is mainly localized on the apical end of parasites by rabbit anti-rBoTRAP2-1 polyclonal serum.
CONCLUSIONS
The rBoTRAP2 could differentiate serum from B. orientalis-infected water buffalo and normal water buffalo, implicating that BoTRAP2 has high immunogenicity and could serve as a candidate antigen for diagnosis of B. orientalis infection in buffalo.
背景
血小板反应蛋白相关的匿名蛋白（TRAP）最初在恶性疟原虫的子孢子中被发现，TRAP 家族蛋白由微线体分泌，并转运至寄生虫表面参与入侵过程。各种 TRAP 蛋白已在顶复门原生动物中被鉴定，但关于东方巴贝斯虫的 TRAP 蛋白的报道较少。
方法
克隆、测序、鉴定并比较了东方巴贝斯虫 TRAP2 的功能域与相关顶复门寄生虫的 TRAP 序列。截短 BoTRAP2 的功能域，命名为 BoTRAP2-1，然后将其克隆到 pET-28a 表达载体中。用免疫三只兔子的方法制备兔抗 rBoTRAP2-1 多克隆抗体。Western blot 分析用于鉴定 BoTRAP2 的天然形式和免疫原性。通过间接荧光测定法（IFA）鉴定 BoTRAP2 的定位。
结果
BoTRAP2 扩增基因长 2817bp，编码约 938aa 的功能域，包含两个 vWFA 结构域、一个 TSP 结构域和一个跨膜结构域。BoTRAP2 的氨基酸序列与牛巴贝斯虫和吉氏巴贝斯虫高度相似。截短的 BoTRAP2-1 的预测三级结构证实，BoTRAP2 包含两个 vWFA 结构域和一个 TSP 结构域，这是蛋白质的主要功能区域。兔抗 rBoTRAP2-1 识别东方巴贝斯虫裂解物中的天然 BoTRAP2。用东方巴贝斯虫感染水牛皮下液的血清反应检测到与 rBoTRAP2-1 相对应的条带，表明该蛋白具有高度的免疫原性。IFA 显示，兔抗 rBoTRAP2-1 多克隆血清主要将 BoTRAP2 定位于寄生虫的顶端。
结论
rBoTRAP2 可区分东方巴贝斯虫感染水牛和正常水牛的血清，提示 BoTRAP2 具有高免疫原性，可作为水牛东方巴贝斯虫感染诊断的候选抗原。
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