is an obligate intracellular apicomplexan parasite， the etiologic agent of neosporosis， and a major cause of reproductive loss in cattle. There is still a lack of effective prevention and treatment measures. The 14-3-3 protein is a widely expressed acidic protein that spontaneously forms dimers within apicomplexan parasites. This protein has been isolated and sequenced in many parasites; however， there are few reports about the 14-3-3 protein. Here， we successfully expressed and purified a recombinant fusion protein of Nc14-3-3 (rNc14-3-3) and prepared a polyclonal antibody. Immunofluorescence and immunogold electron microscopy studies of tachyzoites or -infected cells suggested that 14-3-3 was localiz... More
is an obligate intracellular apicomplexan parasite， the etiologic agent of neosporosis， and a major cause of reproductive loss in cattle. There is still a lack of effective prevention and treatment measures. The 14-3-3 protein is a widely expressed acidic protein that spontaneously forms dimers within apicomplexan parasites. This protein has been isolated and sequenced in many parasites; however， there are few reports about the 14-3-3 protein. Here， we successfully expressed and purified a recombinant fusion protein of Nc14-3-3 (rNc14-3-3) and prepared a polyclonal antibody. Immunofluorescence and immunogold electron microscopy studies of tachyzoites or -infected cells suggested that 14-3-3 was localized in the cytosol and the membrane. Western blotting analysis indicated that rNc14-3-3 could be recognized by -infected mouse sera， suggesting that 14-3-3 may be an infection-associated antigen that is involved in the host immune response. We demonstrated that rNc14-3-3 induced cytokine expression by activating the MAPK and AKT signaling pathways， and inhibitors of p38， ERK， JNK， and AKT could significantly decrease the production of IL-6， IL-12p40， and TNF-α. In addition， phosphorylated nuclear factor-κB (NF-κB/p65) was observed in wild-type peritoneal macrophages (PMs) treated with rNc14-3-3， and the protein level of NF-κB/p65 was reduced in the cytoplasm but increased correspondingly in the nucleus after 2 h of treatment. These results were also observed in deficient in TLR2 PMs. Taken together， our results indicated that the 14-3-3 protein can induce effective immune responses and stimulate cytokine expression by activating the MAPK， AKT， and NF-κB signaling pathways but did not dependent TLR2， suggesting that Nc14-3-3 is a novel vaccine candidate against neosporosis.
