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<p>Abstract</p> <p>Background</p> <p>Toxoplasmosis, caused by an obligate intracellular protozoan parasite <it>Toxoplasma gondii</it>, has been a serious clinical and veterinary problem. Effective DNA vaccines against <it>T. gondii</it> can prevent and control the spread of toxoplasmosis, which is important for both human health and the farming industry. The <it>T. gondii</it> 14-3-3 protein has been proved to be antigenic and immunogenic and was a potential vaccine candidate against toxoplasmosis. In this study, we evaluated the immune responses induced by recombinant plasmids encoding <it>T. gondii</it> surface antigen 1 (SAG1) and 14-3-3 protein by immunizing BALB/c mice intramuscularly.</p> <p>Methods</p> <p>In the present study, BALB/c mice were randomly divided into five groups, including three experimental groups (pSAG1, p14-3-3 and pSAG1/14-3-3) and two control groups (PBS and pBudCE4.1), and were immunized intramuscularly three times. The levels of IgG antibodies and cytokine production in mouse sera were determined by enzyme-linked immunosorbent assays (ELISA). Two weeks after the last immunization, all mice were challenged intraperitoneally (i.p.) with 1×10⁴ tachyzoites of <it>T. gondii</it> and the survival time of mice was observed and recorded every day.</p> <p>Results</p> <p>Mice vaccinated with pSAG1, p14-3-3 or pSAG1/14-3-3 developed high levels of IgG2a and gamma interferon (IFN-γ) and low levels of interleukin-4 (IL-4) and interleukin-10 (IL-10) compared to control groups (PBS or pBudCE4.1), which suggested a modulated Th1 type immune response (P<0.05). After intraperitoneal challenge with 1×10⁴ tachyzoites of <it>T. gondii</it> (RH strain), the survival time of mice in experimental groups was longer than control groups (P<0.05). Mouse immunized with pSAG1/14-3-3 induced a higher level of IgG antibody response and significantly prolonged the survival time when compared with pSAG1 or p14-3-3 (P<0.05).</p> <p>Conclusions</p> <p>The study suggested that <it>T. gondii</it> 14-3-3 protein can induce effective immune responses in BALB/c mice and was a novel DNA vaccine candidate against toxoplasmosis, and the immune protective efficacy elicited by SAG1 gene was also demonstrated. Our results also showed multi-gene vaccine significantly enhanced immune responses and protective efficacy and was superior to the single-gene vaccine.</p>