Study On The Immune Responses Induced By DNA Vaccine With The P30-P22 Of Toxoplasma Gondii In Mice

Toxoplasmosis, a kind of worldwide parasitic zoonoses, occurs in a wide variety of mammals, including humans. The infection of the disease is very common, about more than one third people infected, most infections of adults are asymptomatic. Toxoplasma is an opportunistic protozoan in immunosuppressed and immunodeficient individuals, including patients with organ transplantation, malignant tumor, acquired immune deficiency syndrome (AIDS), toxoplasma can cause them to death. Congenital toxoplasmosis is that toxoplasma crosses the placental barrier from the mother' s blood,which can cause abortions, teratosis and stillbirth. Toxoplasma is able to cause severe disease in animals so it can destroy farming badly . So the manufacture of the vaccine of Toxoplasma gondii is very important in order to prevent and cure toxoplasmosis .In recent years ,with the development of the molecular biology technology , the study on vaccine of Toxoplasma gondii has made great progreses from the earliest dead vaccine to the late nucleic acid vaccine Which has brought about new hope. The surface of tachyzoite is the mainly attached target of host immune system, which has five kinds of major surface antigen including p30, p22, p35, p23 and p43. Among these surface antigens, P30 has both high immunogenicity and immunoreactivity and has no specificity between different strains, so it is most popularlyused nowadays in gene engineering research . The antibody of P30 can kill Toxoplasma in vitro and protect body from infected in vivo. It also can induce IgGs Igfrk IgA antibodies as well asY-interferon and cytotoxic T lymphocyte .The special CD/ T cell of P30 can kill toxoplasma in vitro directly. P22 is another important surface antigen of tachyzoite. According to some reports , P30 and P22 associational antigen has huge effect on mice immune system.In this research , large scale prepared recombinant plasmid pcDNA3.,-P30-P22 was injected into the BALB/c mice via muscle and skin. Two booster injections were given at the 2nd week and 4th week followed the first injection. At the 2nd week, the 4 th week, the 6th week and the 8th week after the last injection, the blood of the mice was taken from the tail and the serum was separated. IgG antibodies , IgG2a and IgGl were detected by ELISA. IFN- Y ^ IL~2 -. IL-4 were detected by sandw ich ELISA .Then muscle, blood, liver and spleen were taken after 4 and 8 weeks immunization to extract tissue DNA as templates and amplify the gene fragments by means of polymerase chain reaction ( PCR ) and identifying the mplifiction products by agarose gel electrophoresis . The proliferation rate of lymophcyte and the killing activity of NK cells were tested by MTT assay , and T cell subgroup CD/, CD8+ were tesed by the indirect immune fluorescent assay. All mice were challenged with highly virulent tachyzoites at the 6th week after the last immunity to observe the survival time. It suggested that the level of the IgG antibodies was remarkably higher than that of control group ,and IgG2a was sightly higher than that of IgGl. The results of IFN-y and IL-2 were higher in the vaccinated groups than that of in control groups and the contents were increased with the vaccinated time prolonged .The survival time of immunized mice was longer than that of conctorl group . The killing rate of the NK cells was remarkably higher than that of control group. The proliferation rate of spleen cell had no difference between the testgroup and the control group. The result of kinetic analysis of CD/ and CD/T cell subgroup showed that the number of CDs* increased and the rate of CD^/CDg* reduced , there was remarkable difference between the control mice and immunized ones. After 4 weeks immunization , P30-P22 gene fragments were amplified out from those tissues of the vaccinated mice which were in accordance with the expected ones. The control group mice could not amplified any fragment. But those specific gene fragments was not amplified from all of the group after 8 weeks.It suggested that mice vaccinated with recombinant pcDNA3.1-P30-P22 plasmid could elicit cellular immune and humoral immune responses so as to making protective immune responses for Toxoplasma gondii infection. It would be a good basis of the research for Toxoplasma gondii vaccine and has a wide application prospect.