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.
Animals ; Cell Line ; Immune Sera/genetics/immunology/*metabolism ; Male ; Mice ; Protozoan Proteins/genetics/*metabolism ; Rabbits ; Recombinant Proteins/immunology ; Sheep ; Sodium-Hydrogen Antiporter/genetics/immunology/*metabolism ; Toxoplasma/genetics/immunology/*metabolism ; Toxoplasmosis/parasitology/prevention & control

Objective: To investigate the expression levels of serum miR-638 in the patients with breast cancer and its clinical value. Methods: One hundred and fifty-two patients with breast cancer were selected as the disease group, and 102 healthy persons as the control group. The expression levels of miR-638 in their serum samples were detected by the quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and the expression levels of serum miR-638 in the patients with different pathological stages, before and after operation, and before and after chemotherapy were compared. The diagnosis efficacy of serum miR-638 alone and in combination with CEA, CA125, CA15-3 for breast cancer was analyzed by the ROC curve and Z test. Results: The expression levels of serum miR-638 in the breast cancer group (3.6 [1.3～10.5]) were significantly lower than that in the control group (79.0 [52.5～120.8],P＜0.01). The linear regression analysis showed that chemotherapy and pathological staging were the main factors influencing the expression levels of serum miR-638. The area under the ROC curve (AUC ROC ) of serum miR-638 in the diagnosis of breast cancer was 0.954. When the cut-off value of serum miR-638 was 27.47, its sensitivity and specificity for the diagnosis of breast cancer were 94% and 86.2%, respectively. The area under the ROC curve (AUC ROC ) of serum miR-638 combined with CEA, CA125 and CA15-3 in the diagnosis of breast cancer was 0.978 8. When the combined cut-off value was 0.29, their sensitivity and specificity for the diagnosis of breast cancer were 95.4% and 89.5%, respectively. There was no significant difference in the AUC ROC between miR-638 alone and combined screening (Z=1.68,P=0.091). Conclusion Serum miR-638 may be a potential molecular marker for the screening of breast cancer.