A Novel Polyclonal Antiserum against Toxoplasma gondii Sodium Hydrogen Exchanger 1.
A Novel Polyclonal Antiserum against Toxoplasma gondii Sodium Hydrogen Exchanger 1.
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一种针对弓形虫弓形氢交换器1的新型多克隆抗血清1。
DOI：
10.3347/kjp.2016.54.1.21
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发表时间：
2016-02
期刊：
影响因子：
--
通讯作者：
Hao W
中科院分区：
文献类型：
--
作者：
Xiao B;Kuang Z;Zhan Y;Chen D;Gao Y;Li M;Luo S;Hao W
钠氢交换器1（NHE 1）广泛分布于细胞质膜，其功能是维持Na+和H+离子的比例。它在pH平衡、细胞增殖、分化、粘附和迁移中起着重要作用。然而，其在弓形虫中的确切亚细胞定位和生物学功能在很大程度上还不清楚。本研究克隆了T.将弓形虫NHE 1（TgNHE 1）的C-末端肽（C-NHE 1）整合到pGEX 4 T-1表达质粒中。基于从免疫表位数据库获得的信息，预测肽序列具有良好的抗原性。异丙基-β-D-硫代半乳糖苷诱导异源基因表达后，成功表达的重组C-NHE 1蛋白的可溶性形式进行纯化谷胱甘肽琼脂糖凝胶珠作为免疫原生产的兔多克隆抗血清。免疫印迹和免疫荧光证实了该抗血清的特异性。该抗血清能降低T.弓形虫侵入宿主细胞，表现为T.在细胞进入过程中与抗血清预孵育的弓形虫。抗血清能降低T.弓形虫体外对宿主细胞的作用，可能是通过阻断Ca 2+的释放。在这方面，这种抗血清有可能成为一个有价值的工具，为进一步研究TgNHE 1。
The sodium hydrogen exchanger 1 (NHE1), which functions in maintaining the ratio of Na+ and H+ ions, is widely distributed in cell plasma membranes. It plays a prominent role in pH balancing, cell proliferation, differentiation, adhesion, and migration. However, its exact subcellular location and biological functions in Toxoplasma gondii are largely unclear. In this study, we cloned the C-terminal sequence of T. gondii NHE1 (TgNHE1) incorporating the C-terminal peptide of NHE1 (C-NHE1) into the pGEX4T-1 expression plasmid. The peptide sequence was predicted to have good antigenicity based on the information obtained from an immune epitope database. After induction of heterologous gene expression with isopropyl-b-D-thiogalactoside, the recombinant C-NHE1 protein successfully expressed in a soluble form was purified by glutathione sepharose beads as an immunogen for production of a rabbit polyclonal antiserum. The specificity of this antiserum was confirmed by western blotting and immunofluorescence. The antiserum could reduce T. gondii invasion into host cells, indicated by the decreased TgNHE1 expression in T. gondii parasites that were pre-incubated with antiserum in the process of cell entry. Furthermore, the antiserum reduced the virulence of T. gondii parasites to host cells in vitro, possibly by blocking the release of Ca2+. In this regard, this antiserum has potential to be a valuable tool for further studies of TgNHE1.