犬新孢子虫中 NcAP2XII-4 的鉴定和功能表征

IF 3 2区 医学 Q1 PARASITOLOGY Parasites & Vectors Pub Date : 2024-09-14 DOI:10.1186/s13071-024-06477-1
Huizhu Nan, Xin Lu, Chao Zhang, Xin Yang, Zhu Ying, Lei Ma
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引用次数: 0

摘要

犬新孢子虫(Neospora caninum)是原生动物寄生虫中的一种,由复杂的信号通路控制。转录调控是调控基因表达的重要途径,但在犬新孢子虫的生命过程中几乎不存在转录调控。然而,迄今为止,对 N. caninum 中 AP2 家族因子转录调控的研究极为有限。之前的一项研究表明,去除 ROPtry 蛋白 5(ROP5)这一重要的毒力因子会导致 N. caninum 中预测的 NcAP2XII-4 的表达水平异常,这表明该因子可能会调控 ROP5 的功能。本研究旨在鉴定 NcAP2XII-4 及其在转录调控中的功能。通过分析 N. caninum 基因组,确定了 NcAP2XII-4 基因。制备并纯化了针对该蛋白的多克隆抗体,并使用免疫印迹(WB)和免疫荧光(IFA)检测了其在寄生虫中的表达和定位。利用CRISPR/Cas9技术在Nc1菌株的基础上构建了ΔNcAP2XII-4菌株,以研究其对犬网虫生长发育的影响,并利用DAP-Seq和电泳迁移分析(EMSA)验证了该基因的转录调控功能。生物信息学分析表明,NcAP2XII-4由11976 bp组成,编码3991个氨基酸,预测分子量为410 kDa。该蛋白有两个 AP2 结构域,分别为 1207aa-1251aa 和 3453aa-3500aa,预计位于细胞核中。PCR、WB和IFA结果与生物信息学分析结果一致。成功构建了ΔNcAP2XII-4,但该菌株无法释放,最终在寄生虫空泡(PVs)中死亡。斑块试验表明,缺乏该基因的寄生虫无法形成斑块。利用 DAP-Seq 技术成功鉴定出了一个基序。成功构建了两种含有 NcAP2XII-4 AP2 结构域的原核表达载体,并制备了 AP2-D1 和 AP2-D2 两种原核表达蛋白以及 ROP5 生物素化探针。通过 EMSA,NcAP2XII-4 与 ROP5 启动子结合,从而调节 ROP5 的转录。NcAP2XII-4 是 N. caninum 的一个重要基因。这项研究为进一步研究犬疫母菌的转录调控奠定了基础,并为开发犬疫母菌疫苗找到了一个新的候选因子。
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Identification and function characterization of NcAP2XII-4 in Neospora caninum
Neospora caninum is a protozoan parasite in the Apicomplexa controlled by complex signaling pathways. Transcriptional control, an important way to regulate gene expression, has been almost absent in the N. caninum life process. However, to date, research on the transcriptional regulation of the AP2 family factors in N. caninum has been extremely limited. A prior study demonstrated that removing rhoptry protein 5 (ROP5), a significant virulence factor, resulted in abnormal expression levels of predicted NcAP2XII-4 in N. caninum, suggesting that the factor may regulate the function of ROP5. This study aimed to identify NcAP2XII-4 and its function in transcriptional regulation. The NcAP2XII-4 gene was identified by analyzing the N. caninum genome. A polyclonal antibody against the protein was prepared and purified, and its expression and localization in the parasite were detected using western blot (WB) and immunofluorescence assay (IFA). The ΔNcAP2XII-4 strain was constructed from the Nc1 strain using CRISPR/Cas9 to study its effect on the growth and development of N. caninum, and DAP-Seq and electrophoretic mobility shift assay (EMSA) were used to verify the transcriptional regulatory functions of the gene. Bioinformatic analysis showed that NcAP2XII-4 consists of 11,976 bp and encodes 3991 amino acids, with a predicted molecular mass of 410 kDa. The protein has two AP2 domains, 1207aa-1251aa and 3453aa-3500aa, and is predicted to be located in the nucleus. The results of PCR, WB, and IFA were in accordance with the bioinformatics analysis. ΔNcAP2XII-4 was successfully constructed, but the strain could not be released and ultimately succumbed within parasitophorous vacuoles (PVs). Plaque assays demonstrated that parasites lacking this gene could not form plaques. One motif was successfully identified using DAP-Seq technique. Two prokaryotic expression vectors containing the AP2 domain of NcAP2XII-4 were successfully constructed, and two prokaryotic expression proteins, AP2-D1 and AP2-D2, and ROP5 biotinylated probes were prepared. Using EMSA, NcAP2XII-4 was shown to regulate ROP5 transcription by binding to its promoter. NcAP2XII-4 is an essential gene in N. caninum. This study provides a foundation for further research on transcriptional regulation in N. caninum and identifies a new candidate factor for the development of vaccines against N. caninum.
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来源期刊
Parasites & Vectors
Parasites & Vectors 医学-寄生虫学
CiteScore
6.30
自引率
9.40%
发文量
433
审稿时长
1.4 months
期刊介绍: Parasites & Vectors is an open access, peer-reviewed online journal dealing with the biology of parasites, parasitic diseases, intermediate hosts, vectors and vector-borne pathogens. Manuscripts published in this journal will be available to all worldwide, with no barriers to access, immediately following acceptance. However, authors retain the copyright of their material and may use it, or distribute it, as they wish. Manuscripts on all aspects of the basic and applied biology of parasites, intermediate hosts, vectors and vector-borne pathogens will be considered. In addition to the traditional and well-established areas of science in these fields, we also aim to provide a vehicle for publication of the rapidly developing resources and technology in parasite, intermediate host and vector genomics and their impacts on biological research. We are able to publish large datasets and extensive results, frequently associated with genomic and post-genomic technologies, which are not readily accommodated in traditional journals. Manuscripts addressing broader issues, for example economics, social sciences and global climate change in relation to parasites, vectors and disease control, are also welcomed.
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