National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro 080-8555, Hokkaido, Japan.
Department of Animal Medicine, Faculty of Veterinary Medicine, South Valley University, Qena City 83523, Qena, Egypt.
Int J Mol Sci. 2022 Mar 12;23(6):3076. doi: 10.3390/ijms23063076.
is a worldwide protozoan parasite that endangers human health and causes enormous economic losses to the animal production sector. A safe and effective vaccine or treatment is needed to reduce these hazards. In this study, we revealed the cyto-nuclear and mitochondrial localization of TgPrx1 and TgPrx3 proteins, respectively. We knocked out the (KO) 1 and 3 genes using a parental type II Prugniaud strain lacking KU80 and HXGPRT genes (PruΔku80Δhxgprt) via CRISPR-Cas9 technology. The successful KO was confirmed using PCR, IFAT, and Western blotting in two clones of both target genes, named KO and KO. Regarding assays, no significant variations between any of the knocked-out clones in KO or KO parasite strains, or even PruΔku80Δhxgprt, were obtained in rates of infection, proliferation, or egress. Nevertheless, mice that were infected with tachyzoites of the KO strain showed a marked decrease in survival rate compared with KO- and PruΔku80Δhxgprt-infected mice. This effect was confirmed using different mouse strains (ICR and C57BL/6J mice), sexes (male and female), and immunological backgrounds (ICR and SCID mice). In addition, KO and KO induced high levels of interferon gamma (IFN-γ) in infected mice at 8 days post infection, and increased IL-6 and IL-12p40 production from murine macrophages cultivated . The results of the present study suggested that can induce anti- immune responses that protect the mice from fatal consequences of toxoplasmosis. The results of our current and previous studies represent as an excellent candidate for sub-unit vaccines, suggesting it may contribute to the control of toxoplasmosis for susceptible humans and animals.
是一种全球性的原生动物寄生虫，危害人类健康，给动物生产部门造成巨大的经济损失。需要一种安全有效的疫苗或治疗方法来降低这些危害。在这项研究中，我们分别揭示了 TgPrx1 和 TgPrx3 蛋白的细胞-核和线粒体定位。我们使用缺乏 KU80 和 HXGPRT 基因的亲本 II 型 Prugniaud 株（PruΔku80Δhxgprt）通过 CRISPR-Cas9 技术敲除了 1 和 3 基因。通过 PCR、IFAT 和 Western blot 在两个靶基因的克隆中证实了成功的 KO，这两个克隆分别命名为 KO 和 KO。关于 测定，在感染率、增殖或逸出方面，任何敲除克隆的 KO 或 KO 寄生虫株，甚至 PruΔku80Δhxgprt，都没有显著差异。然而，用 KO 株的速殖子感染的小鼠与 KO-和 PruΔku80Δhxgprt 感染的小鼠相比，存活率明显下降。这一效应在不同的小鼠品系（ICR 和 C57BL/6J 小鼠）、性别（雄性和雌性）和免疫背景（ICR 和 SCID 小鼠）中得到了证实。此外，KO 和 KO 在感染后 8 天诱导感染小鼠产生高水平的干扰素 γ（IFN-γ），并增加了培养的小鼠巨噬细胞产生 IL-6 和 IL-12p40。本研究的结果表明， 可以诱导抗免疫反应，使小鼠免受弓形虫病致命后果的影响。我们目前和以前的研究结果表明， 作为亚单位疫苗的一个很好的候选者，它可能有助于控制对易感人类和动物的弓形虫病。
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